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1uL of Synthon Mix (mix all three synthons, then take 1 uL)
1uL of Synthon Mix (mix all three synthons, then take 1 uL)
0.5uL Vector

0.5uL Vector

Revision as of 09:12, 23 April 2013
2013 03 06: Designed oligos for PCA1 on CCOMT-1
CCOMT1_Synthon PCA for oligos 1-11, 13-14, 16 (407bp, PCA1_pdt) PCA for oligos 12,15, PCA1_pdt (462bp, PCA2_pdt) Digest PCA2_pdt (417+26+19, EcoRI/BamHI, PCA2_dig) Digest pBca9145-Bca1144 (2967+2958+9, EcoRI/BglII, Vector_dig) Ligate PCA2_dig and Vector_dig (2474, Bca1144-CCOMT1) CCOMT-1_oligo_12: ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCATCGGTCTCCT CCOMT-1_oligo_1: TTATTTGTGTCTCACCTGTAGATCCCATAGGAGACCGATGAGACGATGCAGATCT CCOMT-1_oligo_9: ATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAG CCOMT-1_oligo_3: GAGGCCAACTGCATTGCAAATAAGTTAGCTTCTTCATCAGAAATATGTGTAGGAG CCOMT-1_oligo_5: CTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAA CCOMT-1_oligo_4: CTCTAATAAGTCCAATTCTAAAGCTGATTTCAAAATCATAGGCAAAACTGAAGCA CCOMT-1_oligo_11: ATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGC CCOMT-1_oligo_2: TGAGGCAATTTCGATAGGTGAAATTTGTGCTCCAGGACCTGCCTTAGCAATAAT CCOMT-1_oligo_14: ACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGC CCOMT-1_oligo_6: CCTTAACATCCTATCCAACATTACTGGGGCATCAGGGTTTGTTGTTGGTAATTG CCOMT-1_oligo_7: CCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGAC CCOMT-1_oligo_3: ACCGTCTTGTTGTGTTCTTACAGAGCATGTCAATATAATATAGCAAGCTAATAA CCOMT-1_oligo_10: ATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGC CCOMT-1_oligo_8: ATTCTTAACTAAATATTTTGCTACTGTTGCTAATCCATACAATCTTTGTACCTT CCOMT-1_oligo_16: AACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA CCOMT-1_oligo_15: AAGTATCTTTCCTGTGCCCAGGATCCATGCCGTCTCATAGAAACACCATCTTC -- PCA1_pdt: AGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCA PCA2_pdt: ATATAGATGCCGTCCTAGCGAATTCATGAGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCATGGATCCTGGGCACAGGAAAGATACTT Bca1144-CCOMT1: gAATTCATGAGATCTGCATCGTCTCATCGGTCTCCTATGGGATCTACAGGTGAGACACAAATAACTCCTACACATATTTCTGATGAAGAAGCTAACTTATTTGCAATGCAGTTGGCCTCTGCTTCAGTTTTGCCTATGATTTTGAAATCAGCTTTAGAATTGGACTTATTAGAGATTATTGCTAAGGCAGGTCCTGGAGCACAAATTTCACCTATCGAAATTGCCTCACAATTACCAACAACAAACCCTGATGCCCCAGTAATGTTGGATAGGATGTTAAGGTTATTAGCTTGCTATATTATATTGACATGCTCTGTAAGAACACAACAAGACGGTAAGGTACAAAGATTGTATGGATTAGCAACAGTAGCAAAATATTTAGTTAAGAATGAAGATGGTGTTTCTATGAGACGGCATGGATCCtaaCTCGAGctgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctg
Will perform PCA1 on 2013-03-08
2013 03 08: Did not have PCR plates - will perform PCA1 on CCOMT-1 on 2013-03-09
2013 03 09: Performed PCA1 on CCOMT-1
'''PCA Assembly''' -OK, so you've got a bunch of oligos, now what? First, use this recipe and program to do initial assembly of the oligos (do a separate one of these reactions for each chunk you're assembling): Recipe 38 uL ddH2O 5 ul 10x expand buffer 5 ul 2mM dNTPs 1 ul oligo mixture (100uM total, mixture of oligos after combination of 100uM stocks) 0.75 ul Expand polymerase- From here, sample was given to John Wright: Program (can run JCA/PCA1) 2 min initial denature at 94oC 30 sec denature at 94oC 30 sec anneal at 55oC [This should be the overlap temp of your oligos - vary as needed] 30 sec extension at 72oC repeat 2-4 30 times total
2013 03 12 Regular Zymo Cleanup on PCA1 -> PCA2
Regular Zymo Cleanup The following procedure removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction. It also will remove the buffer and restriction enzymes from a restriction digest reaction. 1) Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. ''ADB kills proteins and allows DNA to stick to the column'' 2) Transfer into the Zymo column (small clear guys) -spin through (1 minute, max g), discard waste. ''DNA is now stuck to white filtered column'' 3) Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) -spin through, discard waste. 4) Add 200 uL of Zymo Wash Buffer -spin through, discard waste. 5) spin for 90 seconds, full speed to dry. 6) elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction -Elute by spinning at max (14000g) for 1 minute -When spinning, face tube caps against direction of spin so cap doesn't break off.
Then perform PCA2:
'''Amplification''' Now, you need to do an amplification of the correct full-length chunks. Clean up the assembly reaction with a zymo column; don't bother running it on a gel - it'll be a smeary mess and won't really help you. Save the purified product in case this step fails! For the amplification reaction, do a normal phusion program with 1 ul of the cleaned up assembly reaction as template, and using the outermost oligos for the chunk. That is: '''Recipe''' 1 ul each outer oligo (10 uM) -Dilute F/R oligos 1:10 from 100uM; in this case, oligo CCOMT1-12/CCOMT1-15 1 ul purified pca product .5 ul phusion 10 ul 5x phusion buffer 5 ul 2mM dNTPs 32.5 ul H2O Samples given to John to run the Program. 1:10 Oligo12/Oligo15 dilutions in small PCR tubes. Also stored purified PCA1 product in 4o box. '''Program''' 2 min initial denature at 94oC 30 sec denature at 94oC 30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product] 30 sec extension at 68oC repeat 2-4 30 times total
2013 03 14 Gel from ALL class samples: File:Http://openwetware.org/images/f/fc/2013 03 13-JCA-gel1.jpg This gel is of the PCA2 products for the various synthons. The first lane is the His-tag part's IPCR product (currently unnamed), then molecular weight standards, then the rest are your PCA2's (I couldn't read the labels). All but three look fine. Lanes 4 and 8 look recoverable with a second PCR. The lane 3's reaction needs to be started over from scratch. Lane 7 just needs a careful gel purification. My sample is one of the last 3 lanes (probably 3rd or 2nd to last
Zymo Cleanup on PCA2 product
1. Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction. 2. Transfer into the Zymo column (small clear guys) 3. spin through, discard waste. 4. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) 5. spin through, discard waste. 6. Add 200 uL of Zymo Wash Buffer 7. spin through, discard waste. 8. spin for 90 seconds, full speed to dry. 9. elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
EcoRI/BamHI Digest on PCA2_zymd
1. 1uL of NEB Buffer 2 (ADD THIS FIRST) 2. 8uL of eluted PCR product 3. 0.5uL EcoRI 4. 0.5uL BamHI Incubate at 37 degrees on the thermocycler for 1hr Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
Zymo Gel Purification
1. Add 2uL Loading Dye to 10uL sample 2. Load all of sample into well -Gel "R" lane 5 3. Run gel @ 180V (Maximum before temperature heats gel to melt) ~20 minutes 4. Cut band out and incubate in 300uL ADB buffer until gel melted 5. Place in Fridge
Stopped here, will finish next week.
- 1 Robert Chen 13:07, 19 March 2013 (EDT)
- 2 Robert Chen 14:10, 21 March 2013 (EDT)
- 3 Robert Chen 13:41, 2 April 2013 (EDT)
- 4 Robert Chen 14:04, 4 April 2013 (EDT)
- 5 Robert Chen 14:06, 9 April 2013 (EDT)
- 6 Robert Chen 14:00, 11 April 2013 (EDT)
- 7 Robert Chen 13:18, 16 April 2013 (EDT)
- 8 Robert Chen 14:04, 18 April 2013 (EDT)
- 9 Robert Chen 16:47, 19 April 2013 (EDT)
- 10 Robert Chen 13:12, 23 April 2013 (EDT)
Robert Chen 13:07, 19 March 2013 (EDT)
Zymo Cleanup after Gel Purification
1. transfer into the Zymo column inside a collection tube (small clear guys) 2. spin through, discard waste. 3. Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol) 4. spin through, discard waste. 5. Add 200 uL of Zymo Wash Buffer 6. spin through, discard waste. 7. spin for 90 seconds, full speed to dry. 8. elute with water into a fresh Eppendorf tube. Water amount should be same as DNA input - '''8uL'''
Robert Chen 14:10, 21 March 2013 (EDT)
Ligation -> Heat Shock -> Plating
Ligation of EcoRI/BamHI digests
6.5uL ddH2O 1uL T4 DNA Ligase Buffer (small red or black-striped tubes) 1uL Vector digest 1uL Insert digest 0.5uL T4 DNA Ligase Pound upside down on the bench to mix Give it a quick spin to send it back to the bottom of the tube Incubate on the benchtop for 30min Put on ice and proceed to the transformation
Transformation Take competent cells to which TSS has been added. Heat shock Rescue with 2YT
Thaw a 200 uL aliquot of cells on ice Add 50 uL of water to the cells (if greater volume is desired) Add 30 uL of KCM to the cells Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution) Add 70 uL of the cell cocktail to the ligation, stir to mix Let sit on ice for 10 min Heat shock for 90 seconds at 42 (longer incubation may work better) Put back on ice for 1 min Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DID NOT RESCUE FOR 1 HOUR BECAUSE AMPICILLIN) Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight
Robert Chen 13:41, 2 April 2013 (EDT)
Plates had 0 colonies on mine. Others had some success. 2/3 of BglII's worked, 1 didn't, suggesting Enzyme is expired? We will redo digest on PCA2 cleaned, and also run a gel on our digested product to make sure we have DNA.
EcoRI/BamHI Digest on PCA2_zymd - Exp 2
1. 1uL of NEB Buffer 2 (ADD THIS FIRST) 2. 8uL of eluted PCR product 3. 0.5uL EcoRI 4. 0.5uL BamHI Incubate at 37 degrees on the thermocycler for 1hr Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
Robert Chen 14:04, 4 April 2013 (EDT)
Remember - this is the second attempt. Our gels showed that our last digest product had DNA, so DNA WAS PRESENT!!
Notice that we didn't add H2O in this procedure to the bacteria!
Ligation EcoRI/BamHI
6.5uL ddH2O 1uL Ligation Buffer 1uL vector 1uL insert 0.5 uL Enzyme (ADD THIS LAST) Incubate 30 minutes on bench after mixing, then put on ice and proceed to transformation
Transformation by Heat Shock
1. Thaw a 200 uL aliquot of cells on ice 2. Add 50 uL of water to the cells (if greater volume is desired) 3. Add 30 uL of KCM to the cells 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution) 5. Add 70 uL of the cell cocktail to the ligation, stir to mix 6. Let sit on ice for 10 min 7. Heat shock for 90 seconds at 42 (longer incubation may work better) 8. Put back on ice for 1 min 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DIDN'T DO THIS) 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight
Robert Chen 14:06, 9 April 2013 (EDT)
We picked colonies without performing colony PCR:
Picking of colonies
For each construct you will pick and later miniprep 2 colonies Add 4mL of LB media with the appropriate antibiotics to a clean test tube Pick a well-isolated, round, and "normal" looking colony with a toothpick Drop it in the test tube Incubate at 37 overnight
Robert Chen 14:00, 11 April 2013 (EDT)
RESULTS: Cultures grew! We now want to: Miniprep cultures, restriction map with ECORI/XhoI, Run on a gel, submit to sequencing
Miniprep Cultures:
1. Miniprep cultures
Miniprep purification of DNA MINIPREP (2mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit) !!!!! Make sure Ethanol has been added to the PE Buffer !!!!! !!!!! Make sure that RNAse has been added to the P1 Buffer !!!!! Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. Dump supernatant Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake. Spin in centrifuge at top speed for 5 minutes. Label blue columns with an alcohol-resistant lab pen. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin) Wash each column with 500 uL of PB buffer. Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again. Wash with 750uL of PE buffer (washes the salts off the resins). Spin in centrifuge at full speed for 15 seconds and flick out liquid again. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol. Label new Microcentrifuge tubes and put columns in them. Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides). Spin in centrifuge at top speed for 30 seconds. Take out columns and cap the tubes. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.
Test Digest with EcoRI and XhoI
7 uL ddH2O 1uL Miniprepped plasmid 1uL 10x NEB Buffer 2 0.5uL EcoRI 0.5uL BamHI Incubate at 37 on the thermocycler for 30 minutes Run an analytical gel
Take a picture of the gel Calculate the expected fragment sizes Are the calculated sizes consistent with the bands on the gel? Other notes about Test Digest
In this protocol, you are trying to 'map' your plasmid to determine whether it is likely to be the desired sequence. There are two pieces of information you need to consider before setting up the digest: How much DNA should I use? What restriction enzymes should I use? The choice of how much of your miniprepped plasmid to add depends on the concentration of DNA in the sample, which primarily reflects the copy number of the plasmid in the cell, which is primarily determined by what origin of replication is on the plasmid. However, subtleties about how you do the miniprep procedure, what else is on the plasmid, and any stress/load present in the bacterium can affect the plasmid yield. To a first approximation, you can make this decision based on the origin of replication: colE1 (pUC plasmids, such as pBca9145, pBca9523, pBca9525 vectors) : use 1uL as it is very high copy colE1+ROP (pBR322-derived plasmids that retain ROP) : use 2uL as it's a little lower when ROP is present p15A (such as pBgl0001 and pAC vectors) : use 4uL as it is medium copy pSC101 : use 6 uL as it's lower copy F plasmid (such as BACs) : use 8uL since these are very low copy Just use that volume of DNA, and adjust the amount of water so that things add up to 10uL The choice of what restriction enzymes to use involves using ApE (or an equivalent tool that predicts the fragmentation pattern of a restriction digest). Your primary goal is to distinguish your product from the plasmid that you inserted your part into. Often that means, say, distinguishing pBca9145-Bca1144#5 from your product pBca9145-sbb1222. The reason for this is that primarily what bleeds through onto the plate is this parent plasmid, or mutants thereof. So, you want to find one or two enzymes that gives a clearly-different restriction pattern in your product from that of the parent vector. Scenarios: A good place to start is considering using combinations of the BioBrick format enzymes (for BglBricks, that means EcoRI, BamHI, BglII, XhoI) or the enzymes you used during your cloning. If the part that you inserted is significantly larger or smaller than the one in the parent vector, this is a very good strategy as you'll get 2 bands for both parent and product, and you can tell by the size which is which. Sometimes your part is too similar in size to the parent part. Under these circumstances, look for a restriction site that is in the product part but absent in the parent part. Use that enzyme in combination with one of the BioBrick enzymes. Sometimes your part is too short to see well on the gel, even though it is readily distinguishable from the parent part. Here, you should consider using other enzymes in the backbone of both plasmids in conjunction with a BioBrick enzyme to get a unique pattern. AlwNI is often useful in this regard. 'fingerprinting' the plasmids with DpnI often will disambiguate hard-to-distinguish cases. Here, you use an enzyme that cuts many times in the plasmids, and the fragmentation pattern will often give a different spectrum in the parent and product.
Sent ? and ? for sequencing
Robert Chen 13:18, 16 April 2013 (EDT)
Sequencing worked!
Check sequencing against PCA2 product from beginning, CONFIRMED! Chose CCOMT1A (Sequencing A5). We can't continue because CCOMT2 doesn't have a sequence we can use.
Finished for the day.
Robert Chen 14:04, 18 April 2013 (EDT)
Helping out with CCOMT2 so that we can get our project up to speed!
1. Mini-prep CCOMT2 2. Restriction Map with BsmBI (Eco/Xho were missing):
6.5uL H2O 2uL DNA 1uL NEB Buffer 3 0.5uL BsmBI Thermo for 30 minutes - 1 hour Load gel (all into 6x diluted Dye
3. Send to sequencing!
Robert Chen 16:47, 19 April 2013 (EDT)
Golden Gate Reaction on CCOMT1,2,3
6.5uL ddH2O 1uL Ligase Buffer 0.5uL Ligase 0.5uL BsmB1 1uL of Synthon Mix (mix all three synthons, then take 1 uL) 0.5uL Vector
Robert Chen 13:12, 23 April 2013 (EDT)
To do: Transform Cells with Golden Gate Product, Rescue (Chlorinphenicol is a translation inhibitor, so we need a rescue step to allow cells to start producing the antibiotic resistance), and Plate
Heat Shock Transformation, Rescue, and Plating
1. Thaw a 200 uL aliquot of cells on ice 2. Add 50 uL of water to the cells (if greater volume is desired) 3. Add '''20 uL''' of KCM to the cells 4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution) 5. Add 70 uL of the cell cocktail to the ligation, stir to mix 6. Let sit on ice for 10 min 7. Heat shock for 90 seconds at 42 (longer incubation may work better) 8. Put back on ice for 1 min 9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour 10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight


text:
Sexual Problems: Questions About Your Sexual HistorySkip to the navigation
Topic Overview
Women often begin the process of diagnosing sexual dysfunction by noticing an absence of sexual desire or satisfaction. Your health professional will work with you to identify your symptoms and the history of those symptoms. The answers to the following questions may be helpful to you and your health professional in finding out the cause of your sexual problem and designing a treatment plan.
- Are you currently sexually active?
- If so, are you sexually active with men, women, or both?
- Are you or your partner experiencing sexual difficulties?
- Have you noticed a change in your sexual activity?
- Are you satisfied with the quality and frequency of sexual activity?
- Have you ever experienced any unwanted touching or sexual activity?
- What are your expectations and goals for treatment?
Other questions that may be helpful in your diagnosis include the following:
- Are you having difficulty initiating sexual activity?
- Are you having difficulty becoming aroused when you want to be sexual?
- Do you experience as much arousal as you expect or would like?
- Do you experience dryness in your vagina during intercourse?
- Do you have pain during intercourse?
If answering these questions leads you to identify a sexual problem, the following questions may provide helpful additional information.
- Is this a new problem, or has it always been present?
- Have you had this problem with all of your partners?
- Does anything make it better or worse?
- How much of a concern is this for you and your partner?
- Do you have any idea what may have caused your sexual problem?
- Have you received any treatment for this problem?
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ByHealthwise Staff
Primary Medical Reviewer Sarah Marshall, MD - Family Medicine
Specialist Medical Reviewer Martin J. Gabica, MD - Family Medicine
Current as ofOctober 13, 2016
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Current as of: October 13, 2016
Author: Healthwise Staff
Medical Review: Sarah Marshall, MD - Family Medicine & Martin J. Gabica, MD - Family Medicine

subset: commoncrawl


text: Doug Smith's blog mentioned that BC, Stefanski and the rest of the team are at the combine and have spoken with Shabazz Mohammed. Now if he was available with the 12th pick I may reconsider the Lowry trade. There has been mention of Shabazz' stock dropping due to attitude problem, and also about lieing about his age, so perhaps he could drop to 12 or even lower. I don't know much about him, but he seems like a natural scorer.


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IRVING, TX -- In its 2012 third-quarter report, CEC Entertainment Inc., owner of the Chuck E. Cheese's chain of restaurant-arcades, said profits dropped by one-third, from $11.7 million to $7.8 million, from July 1 through Sept. 30. Comparable stores sales fell 2.3% for the quarter.
Total revenues for the third quarter decreased 1.7%, or $3.4 million, to $196.6 million from $200 million for the third quarter of 2011. Company officials said the decline was partially offset by additional revenues from six new stores opened or acquired since the end of the third quarter a year ago.
CEC president and chief executive Michael Magusiak said the company saw decreased sales because it made excessively steep cuts in TV advertising aimed at children.
Beginning last February, CEC announced a new marketing strategy that would target mothers with radio spots. | SEE STORY
In May, CEC followed up by announcing an aggressive effort to leverage social media, again targeting mothers. | SEE STORY
Local news media said a Dallas-based advertising firm, the Richards Group, is working with CEC to revise its marketing campaign. Magusiak said CEC would "reverse" its earlier decision to de-emphasize TV and would spend 25% more on media in 2013 to create a stronger branding and value message focused on lower prices and coupons.
He added that CEC would spend $41 million to upgrade 123 existing stores. Of these, 25 will be expanded, five will be extensively remodeled and 93 will get more new games.
Magusiak declined to make any forecasts about company performance for the fourth quarter of 2012. He cited uncertainty regarding fourth-quarter sales, "including the ultimate impact of Superstorm Sandy in our lowest seasonal quarter."
Magusiak said that October sales were strong. He also said the company expects its revamped marketing campaign, new pricing and investment will combine to create a stronger performance in 2013.
Marking its 35th anniversary this year, CEC and its franchisees operate 562 Chuck E. Cheese's stores located in 48 states, and in eight countries or territories.
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During the New York Knicks victory over the Chicago Bulls, head coach Mike Woodson chided Smith for his poor shot selection, according to the New York Daily News[Only registered and activated users can see links. ] Frank Isola. Never one to shy away from confrontation, Smith reportedly had some words of his own for Woodson
Good move, Swish. Argue with Coach Woody after he's defended you more than anyone else these last two seasons. Good move.According to a team source, Woodson admonished Smith for poor shot selection during the Bulls game and then became upset over something Smith said on the bench. Woodson waited until after the game to express his disappointment with Smiths attitude and reminded the volatile shooting guard that hes been his strongest advocate for the past two seasons."Mike said that after all Ive done for you I cant have you talking back to me like that," the source claimed. The same source added that Smith made a lewd comment that angered Woodson.
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I've got nothing to say other than JR needs to go.


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According to Alexander Furnas writing at The Atlantic, our ever-inventive friends at the Department of Homeland Security and the TSA are ever on the trail of new and more intrusive ways to make travel even more unpleasant. They have developed a 'pre-crime' detection program (reminiscent of the film Minority Report) known as FAST (Future Attribute Screening Technology), because all bad programs need a catchy acronym to help gain acceptance.
FAST will remotely monitor physiological and behavioral cues, like elevated heart rate, eye movement, body temperature, facial patterns, and body language, and analyze these cues algorithmically for statistical aberrance in an attempt to identify people with nefarious intentions.
This all sounds super-scientific and precise.The problem is that whenever you are trying to determine the likelihood of a low-probability event with anything other than a 100% accurate predictive test, you encounter the inevitable problem of false positives.
I have written before about the false-positive problem as applied to medical diagnostic tests that can cause unnecessary fear and alarm to patients who are unaware of how to properly interpret statistical data. This same false-positive problem will inevitably overwhelm the extremely intrusive and privacy invading FAST program. Furnas runs some numbers:
Here is why: let's assume for a moment that 1 in 1,000,000 people is a terrorist about to commit a crime. Terrorists are actually probably much much more rare, or we would have a whole lot more acts of terrorism, given the daily throughput of the global transportation system. Now lets imagine the FAST algorithm correctly classifies 99.99 percent of observations — an incredibly high rate of accuracy for any big data-based predictive model. Even with this unbelievable level of accuracy, the system would still falsely accuse 99 people of being terrorists for every one terrorist it finds. Given that none of these people would have actually committed a terrorist act yet distinguishing the innocent false positives from the guilty might be a non-trivial, and invasive task.
This is why I think that once we have locked cockpit doors, installed metal detectors, and passengers feel free to intervene to overpower aggressive fellow passengers, we have reached the level of reasonable precautions, in that there is little or no chance of an airplane being used as a weapon and the chances of dying in a hijacked plane are far less than in everyday life. We are now well past the stage where diminishing returns have set in and all we are doing is making air travel infuriating and discriminatory.
I wrote before about the debate between Sam Harris and Bruce Schneier, where the latter challenged the former's support for the profiling of Muslims. The entire exchange can be read here. After giving all the technical reasons as to why the absurdly expensive and intrusive methods adopted by the TSA and the Department of Homeland Security are undesirable, Schneier's conclusion is worth repeating:
But perhaps most importantly, we should refuse to be terrorized. Terrorism isn't really a crime against people or property; it's a crime against our minds. If we are terrorized, then the terrorists win even if their plots fail. If we refuse to be terrorized, then the terrorists lose even if their plots succeed.
We are at a small risk every day from death or injury due to criminals, maniacs, accidents, and defective products. We have learned to live with the fact that we cannot have perfect protection from such threats without making life intolerable. Similarly there is no question that there will be a small risk of terrorist attacks of varying levels of effectiveness as long as we continue to invade other countries and kill their people using drones and other means. Why cannot we live with the same tradeoffs of safety versus liberty when the source of danger is terrorism?
Stephen M. Walt recently attended a conference in Turkey and describes his own traveling experience:
Finally, I flew here on Turkish Airlines via John F. Kennedy Airport in New York. The flight was fine, but the on-the-ground experience in JFK was one of the more miserable I've had in the past decade. And I couldn't help but wonder — and not for the first time — how this affects how non-Americans view the U.S. when they arrive here. So I have the following modest proposal to offer: Every U.S. congressperson should be forced to fly through JFK on their own (i.e., with no staff to help), and to go through the normal TSA procedure (no VIP lines). And then they should be flown to a really first class airport in some foreign country (say, in Singapore, or Munich), so that they can see just how decrepit U.S. transportation infrastructure has become. And a few hours interacting with the Keystone Cops at JFK's TSA checkpoints would be instructive for them too.
I think Americans have no idea how rapidly this country's public services and infrastructure, once the envy of the world, is deteriorating as massive amounts of resources are diverted to defense and tax cuts for the wealthy. Add to that the needless aggravation caused by the never-ending paranoia generated by the so-called 'war on terror' and you have a country that people will be reluctant to come to unless they have to.


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MISSION VIEJO – Ceramic sculptures, illustrations and paintings were on display during Capistrano Valley High School's annual Art Gallery Gala on Wednesday.
School staff, students, family and members of the Mission Viejo community attended the event to show their appreciation of the students' artwork and see some of their live performances.
Attendees were given a preview of Shakespeare's "The Taming of the Shrew", which the students will perform Friday and Saturday.


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ORICA-GreenEDGE's Michael Albasini made it two for two at the Tour de Romandie, taking out the second road stage of his home country tour. A nearly 70-strong bunch contested the sprint in Montreux where Albasini beat out Tony Hurel (Europcar) and Giacomo Nizzolo (Trek Factory Racing) to the line. With the stage two win came ten bonus seconds and the yellow overall leader's jersey.
"My teammates put me under pressure today," said Albasini with a laugh. "I was not feeling great today. I had the race from yesterday in my legs. When we started riding on the front, I knew I had to deliver something because they did a really big effort. I'm happy to win after the work they did today."
After his brilliant victory yesterday, Albasini was the protected rider for the Australian outfit on stage two. Stephens expected a reduced bunch to contest the finish and was confident the stage one winner would be in the mix at the finish.
"Maybe it was a smart move to put me under pressure," said Albasini. "I had to do something with the way they rode. I'm really thankful for the work they did."
Martin Kohler (BMC) and Pirmin Lang (IAM) animated the early action. The duo broke away from the bunch shortly after the stage start in Sion. In the opening hour, the leaders built up a maximum advantage of more than 13 minutes.
As the breakaway's advantage continued to fall, a handful other teams committed to the chase. Belkin and Omega Pharma – Quick-Step sent riders to the front. Thirty kilometres from the finish, the peloton had crested the uncategorized climb that came quick on the heels of the second category three mountain, and the breakaway's advantage had fallen below the two minute mark.
The peloton continued to chase on the downhill run-in to the finish, overtaking Kohler and Lang inside the final three kilometres. Light rain and one short steep kick before the final plunge toward the line made for a technical finale. Overnight race leader Michal Kwiatkowski was the first to accelerate to the line, but there was no stopping Albasini once he opened his sprint.
"When you're not feeling good, you never know how the sprint is going to work out," said Albasini. "I started moving up really late. I left it until three or four kilometres to go. I watched out for some quick wheels, and I was lucky to choose the right one. I stayed on the wheel up to 100 metres when I could go just on the right side. I was quite fresh because I wasn't catching any wind. It was perfect for me. I was surprised to win such a big bunch sprint
While Albasini will wear the yellow jersey during the queen stage of the Tour de Romandie, the team's efforts will focus on Meyer. The Australian is better equipped to contend with the four first category climbs in the mountains behind Aigle.
"The stage is something nobody can take away," said Albasini. "It's a bit different with the yellow jersey, but once you have it, you're never going to give it away for free. I'm going to fight tomorrow and try to stay there as long as possible."
"Unless something happens where there is a drastic change to the race route, which is the way he won the overall in Catalunya two years ago, Michael doesn't have much of a chance to win the Tour of Switerland," Stephens explained. "He won't go down without a fight, but it's unrealistic to think he's going to get over the big mountains with the best guys in the world. Our best chance for a good result tomorrow is in Cameron Meyer, and we'll focus on him."


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Another "when will Rex go" or "what will he need to do to keep his job" thread and the usual moron parade shows up lead by the county idiot.
Throw in an occasional new theory...he was set up to quit but wants the money. Can't get any dumber than that one.


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Two Florida-breds could factor prominently in Grade 1 races today on opposite ends of the country. On the East Cost, Unlimited Budget is the second choice to win the Coaching Club American Oaks at Saratoga Race Course in New York.
Two Florida-breds could factor prominently in Grade 1 races today on opposite ends of the country.
On the East Cost, Unlimited Budget is the second choice to win the Coaching Club American Oaks at Saratoga Race Course in New York.
Meanwhile, out west, Jeranimo is the favorite in the Eddie Read Stakes at Del Mar in California.
Unlimited Budget was bred in Marion County by Ocala Stud farm. The daughter of Street Sense was undefeated in her career entering the Kentucky Oaks at Churchill Downs in May. Despite getting bumped at the start of the race, she was able to finish third.
She came back in June and tested the males in the Belmont Stakes, where she finished a game sixth in the 1 1/2-mile race.
Today she is the 2-1 favorite in the 1 1/8-mile Coaching Club worth $300,000. The race only drew five entries, but sets up a rematch between Unlimited Budget and Princess of Sylmar, who won the Kentucky Oaks.
Princess of Sylmar, a daughter of Majestic Warrior, is the 9-5 favorite. She breaks from the No. 4 posts, right next to Unlimited Budget in post No. 3.
Unlimited Budget went through Ocala Stud's training program and was sold at the Ocala Breeders' Sales 2012 March auction for $475,000. She was purchased by Repole Stables, which is owned by Vitaminwater founder Mike Repole.
Fellow Florida-bred Marathon Lady is a 5-1 choice in the race. Bred by Stud El Aguila, she is owned by Alex and JoAnn Lieblong. A daughter of Winding Oaks stud Graeme Hall, she has hit the board in six of her seven career races and finished third in the Grade 1 Mother Goose Stakes at Belmont on June 22.
The Coaching Club is scheduled for 5:45 p.m. and is race No. 10.
Jeranimo hasn't won a race since January, when he took the Grade 2 San Gabriel Stakes, but he is the favorite in today's Grade 1 Eddie Read Stakes at Del Mar.
In his last start, the 7-year-old son of Congaree, finished fourth in the Grade 1 Shoemaker Mile Stakes at Hollywood Park in California in June. He was defending his 2012 title, but showed no rally in the 2013 edition.
The $300,000 Eddie Read drew 10 entries for the 1 1/8-mile turf race. Listed at odds of 7-2, he will be ridden by Rafael Bejarano and breaks from the No. 4 post.
Jeranimo is a multiple graded stakes winner and has earned more than $1.2 million in his career. Bred by Brylynn Farm, he is owned by Robert LaPenta and B.J. Wright.
Japanese-bred Barocci is the second choice at odds of 9-2. The 5-year-old son of Deep Impact has not won a stakes race since coming to North America from Europe in 2012. He won his last race, an allowance at Hollywood Park on July 5.
The Eddie Read is scheduled for 8:42 p.m. and is race No. 8.


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By Rob Mahoney
The Internet's secondary market offers a chance to snatch up all manner of NBA gems -- from autographed memorabilia to championship rings to, apparently, the entire hardwood court of a relocated team. According to this Craigslist post, the new owners of the Vancouver Grizzlies' former practice facility are seeking someone to take the entire hardwood floor off their hands. It's hardly uncommon for a swath of NBA court to pop up on eBay or the like, but seeing the entire plot for sale in one place is just a bit more rare, and far more valuable for the collector in need of a stocking stuffer spanning some 4,800 square feet.
If you happen to have $13,000 burning a hole in your pocket and have a Canadian friend who loves basketball, appreciates quintessentially '90s NBA color schemes, hasn't disowned the Grizz outright since their departure from the country, didn't turn coat to pledge allegiance to the Raptors and by chance owns an empty warehouse or hangar in which to store a full-size NBA court, then you just might be in a position to pick up this gem and make both the seller and the recipient very happy.J.E. Skeets @NBAVancouver


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The Benefit of Poultices During the Show Season
- Jun 21, 2011
- Topics:
Poultice can help reduce tissue heat and swelling caused by the demands of high intensity training and performance.
Photo: Anne M. Eberhardt/The Horse
During the competition season horses are generally worked harder, stalled more frequently, and are transported more often. This combination can generate a greater amount of stress on the horse's muscles and connective tissue. Jack Grogan, CN, chief science officer at Uckele Health & Nutrition, relays that it is vitally important that the strong, yet delicate leg muscles and connective tissue be protected and cared for day in and day out, especially during show season.
"As a result of the increased work load, it is very common for muscles, tendons, ligaments, and connective tissue to be overworked or strained, not necessarily to the point of injury, but as a normal consequence of the extra training and performance," he explains.
Grogan emphasizes that the muscles and connective tissues of the legs require rest and care to recuperate as completely as possible to perform at the highest level of the horse's physical potential: "One of the first lines of defense to protect the muscles, tendons, and ligaments from the stress of training and performing is a high quality poultice. This functions as a drawing substance, which soothes and comforts the tissues. A good poultice reduces tissue heat and swelling caused by the connective tissue stress that occurs from the substantially greater work load and increased demands of high intensity training and performance."
Grogan recommends a clay-based formula to provide a temporary cooling benefit for inflamed tissues and natural astringent properties, which can support a faster recuperation. He points out that when it comes to injury, a good rule of thumb is cold for fresh trauma and as a preventive after heavy work.
"In the early acute phases after injury, cold minimizes the aggressive stress response to the injury, and can reduce pain," Grogan says, adding that by reducing the initial stress of the injury, the risk of further, more damaging injury from swelling and inflammation is minimized.
In addition, Grogan points out that poultices can benefit all horses, not just performance horses, supporting and soothing the legs, muscles, and connective tissue. A high quality poultice can reduce pain or discomfort from normal daily exercise and activity, relieve soreness and stiffness in muscles and joints after a hard workout, and provide natural astringent properties.
"While ice is the preferred cold therapy, it's not always a convenient option," Grogan concludes, "Poultices are an excellent alternative option to cool things down and are frequently used as cold therapy on the lower legs (tendons, ligaments, joints) and hooves (traumatic injury, bruises, abscesses) to reduce swelling and aid healing."
About the Author
Uckele Health and Nutrition
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Managing Neck and Back Pain in Sport Horses


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December is here and Christmas is less than three weeks away. (We are scrambling to get our holiday shopping done, too.) As you and your book club near your final gathering for 2013, we'd like to share some of our favorite books and literary pairings for readers of all kinds!
If you are giving the gift of Fannie Flagg this season then we recommend a photo album. This is a fitting and timeless gift to accompany this bestselling novel's generation-spanning storyline.
A teapot is a central symbol in Tell the Wolves I'm Home, so pairing one with the book and some holiday teas will warm up any gift (Hint: We like chai and apple cinnamon this time of year!)
And finally, we recommend bundling The Wishing Thread, an enchanting debut novel, with yarn and a pair of knitting needles. A perfect read for book lovers and knitters alike!
What other literary-themed gift ideas do you have? Share with us on Facebook and Twitter!


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Thanksgiving looms large, and it strikes dread (or at least boredom) into the heart of many a wine writer. Lord, not another paean to pinot!
Not me. I've attended enough autumnal spreads through the years to know that almost any wine will find a welcome food companion at a typical American Thanksgiving feast. Even the much-maligned turkey, in recent times, has become the object of gastronomic experimentation. The spicy bird I tried a few years ago with chopped jalapenos in the stuffing and soaked in a garlic-and-citrus-flavored marinade was made for a big Paso Robles zinfandel.
So, in that freewheeling spirit, here are a few wines I've tried recently that will delight most palates and pair with a lot of things on the table, whether your Thanksgiving meal is traditional, eclectic or fashion-forward. This week, we'll tackle the bubblies and whites; next week, the reds.
J Sparkling Brut Rosé (under $30)
What better way to kick off a holiday feast than with a glass of rose-colored sparkling wine? One of my favorite California bubbly houses makes a great rosé. It has been singled out by the Connoisseur's Guide to California Wine: "This frothy, crisply balanced bubbly places the accent on freshness and grace even while showing a trace of proper rosé grip. It ends with a slight lemony lift at the finish, yet its fix on fruit is tenacious." It's worth the trip to the winery, too: J's Healdsburg tasting room is one of the friendliest in the state.
Chateau Ste. Michelle Dry Riesling 2010 (under $9)
I'm a huge fan of this large but often overlooked Washington winery. The Chateau Ste. Michelle Dry Riesling is refreshing, dry and crisp. You'll get stone-fruit flavors such as peach along with floral hints, solid but not overwhelming acidity, and a finish that's surprisingly long and elegant. It exudes beautiful white peach and floral aromas. Wine.com: "This is an incredibly versatile food wine and a favorite with oysters." One of the best white wine bargains around; it's a natural with light appetizers.
Conde de Valdemar 'Finca Alto Cantabria' Barrel-fermented White 2009 (around $14)
We fell in love instantly with this unusual wine, made from Viura grapes planted in 1970. Producer Bodegas Valdemar claims it's the first barrel-fermented white wine from Rioja. It spent four months in new French oak, which gives it wonderful floral aromatics and an undertone some have described as apple peel. The palate is lively, fresh and shot through with complex fruit; we also enjoyed the slight zing of spice on an otherwise clean finish. It would pair well with fish or mildly seasoned poultry.
Byron Santa Maria Valley Chardonnay 2010
(under $19)
Winemaker "Byron" Ken Brown's big, luscious and inexpensive chardonnay, which achieved cult status in the mid-1980s, helped get me hooked on California wine. He moved on long ago, but I tasted Byron Chardonnay again last summer after avoiding it for years, and it was like taking a trip in the way-back machine. The old sorcery is still there under winemaker Jonathan Nagy, and at affordable prices, too: Byron charges about $30 for its high-end Nielsen Vineyard Chardonnay and $35 for its pinot noir. Wine Enthusiast seconds my regard for this well-priced chardonnay: "Rich, spicy and oaky, it exudes a honeyed quality along with pineapple and lemon meringue notes. The brisk Santa Maria acidity provides clean, mouthwatering balance. Pair this opulent Chardonnay with lobster, shrimp and salmon."
Contact the writer: 714-796-7979 or email@example.com
Contact the writer: 714-796-7979 or email@example.com


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The Angel of Fredericksburg
Gen. J. B. Kershaw, of South Carolina, published some years ago a deeply interesting narrative concerning "Richard Kirkland, the humane hero of Fredericksburg," a sergeant in the Second South Carolina regiment. After the bloody repulse of the Federals at Fredericksburg, near the foot of Marye's hill, they left their many killed and wounded lying between the lines, and the piteous cries of the brave men on account of pain and thirst appealed to the sympathies of the soldiers of both armies. Kirkland went to General Kershaw, who was then in command of the Confederates at that point, and said with deep emotion: "General, I can't stand this." "What is the matter, Sergeant?" asked the general. He replied: "All day I have heard those poor people crying for water and I can stand it no longer. I come to ask permission to go and give them water." The general regarded him for a moment with feelings of profound admiration, and said: "Kirkland, don't you know that you would get a bullet through your head the moment you stepped over the wall? .... Yes," he said, "I know that I may, but if you will let me, I am willing to try it." After a pause the general said: "Kirkland, I ought not to allow you to run such a risk, but the sentiment which actuates you is so noble that I will not refuse your request, trusting that God may protect you. You may go." With light heart and buoyant step the humane hero, armed with all of the canteens he could carry filled with water, crossed the wall, went unharmed through the shower of bullets which at first greeted him, and reached and relieved the nearest sufferer, pouring down his parched throat the life-giving fluid, putting him in a more comfortable position, and leaving him a canteen filled with water. His purpose now being apparent, the Federals ceased to fire on him, and for an hour and a half, amid the plaudits of both armies, this angel of mercy went on his mission from man to man of the wounded enemy--his comrades gladly filling his canteens for him and being prevented from joining him in his labor of love only by the orders against their crossing the line--until all on that part of the field were relieved. It needs only to be added--since "the bravest are the tenderest and the loving are the daring"--that Sergeant Kirkland so greatly distinguished himself at Gettysburg that he was promoted for "conspicuous gallantry," and that he fell on the victorious field of Chickamauga, bravely doing his duty. But he will be known in the annals of the war as "The humane hero of Fredericksburg," and as he had but a short time before found "Christ in the camp," I doubt not that he wears now a bright crown bestowed by Him who promises that a cup of cold water given in the right spirit shall not lose its reward.
Source: "Confederate Military History," Volume 12
Richard Kirkland, The Humane Hero of Fredericksburg General Kershaw tells the story of this young hero at Fredericksburg
This page last updated 11/25/06


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Tiger-Cats rally past Alouettes
The Hamilton Tiger-Cats put up 17 points in the fourth quarter on Saturday afternoon to rally for a 27-24 win over the Montreal Alouettes.
The Tiger-Cats failed to cover the 5.5-point spread in this contest, while the combined score fell UNDER the day's posted total (53).
Troy Smith threw two touchdown passes and two interceptions in the losing cause for the Alouettes.


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A news item today talks about efforts by Christian evangelists to boost their witnessing in such New England states as Vermont, New Hampshire, Maine and Massachusetts, an area of the country that has become even more secular than the Pacific Northwest. Reportedly, up to 22% of New England residents claim no religious faith of any kind. This is absolutely wonderful news of a trend that I hope continues to spread. But it's sad faces all around for the poor folks at places like Redeemer Fellowship Church.
Several Christian denominations see New England as a "mission field" — a term often associated with unchurched, foreign lands. As they evangelize and work to plant new churches, they speak of possibility, but also frustration. The area's highly educated population is skeptical and often indifferent to their faith.
"About once every hour, I give up. It's tough, man," said a half-joking Joe Souza, a Southern Baptist missionary working north of Boston. "It's like, you found a cure for cancer and you want to give it away and nobody wants it."
That last remark illustrates with blazing clarity why fellows like Souza do not understand their difficulty in winning converts. Read the preceding sentence, Joe, where the people whom you are trying to reach are described as "highly educated" and "skeptical." There's your problem. You are dealing with people who are sufficiently intelligent to realize, much moreso than you, that you emphatically do not have anything to offer that can be remotely likened to a cure for cancer. (For one thing, if Souza's religion really were both true and as good as a cure for cancer, then there would be a cure for cancer. All powerful magic sky deity, remember?)
No, Christianity is in fact peddling an inferior product, one that offers the empty solace of "faith" in response to real-world problems, and then, with staggering arrogance, turns around and threatens people with eternal torture for non-compliance. Maybe there's a placebo effect in Christianity that can, I suppose, be argued to have better benefit than no effect at all. But "it's better than nothing" is not exactly what you'd call a ringing endorsement for a belief system that treats the human intellect as if it's something that can be won over with carrot (Heaven) and stick (Hell) theology.
The work is slow and its fruits can be scarce. Souza said people are generally polite, even interested in talking about spiritual matters. But they don't hesitate to reject invitations. He recalled a man with whom he recently shared his faith at the mall courteously declining to even take a card.
Of course. This man realized that Souza had nothing to offer that he wanted or needed. This is always how people respond to empty sales pitches: with indifference. Oh, it's a telemarketer offering me a limited-time-only deal on subscriptions to magazines I don't even read in the first place? Gee, why am I not clamoring to take advantage of that? Golly, it's a smiling but empty-headed buffoon in the food court telling me all about how much his invisible friend loves me? Sure, great, whatever — can you pass the sugar?
This trend, I think, is a terrific riposte to claims that those who criticize the "New Atheists" often make: that we may as well accommodate religion, because it is such a thoroughly ingrained part of our cultural landscape that we'll never be rid of it. Clearly, as the increasingly educated and skeptical population of New England are demonstrating, that is untrue. It doesn't take much to divest yourself of inferior products in your life. You just need to become a smart shopper.


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By Date
U.S. EPA Fines Reno Area Facility $79,500 for Hazardous Waste Violations
Release Date: 11/30/2011
Contact Information: Margot Perez-Sullivan, firstname.lastname@example.com
SAN FRANCISCO—The U.S. Environmental Protection Agency fined a hazardous waste treatment, storage and recycling company $79,500 for the improper management of hazardous waste at its Fernley facility.
A 2010 inspection at 21st Century Environmental Management of Nevada, LLC, uncovered violations of federal standards governing the handling and storage of hazardous waste. In this case, the facility processes and recycles a variety of hazardous materials. Among the violations were storage of hazardous waste in an unpermitted area, cracked and deteriorated containment areas, and a leaking container.
Under the EPA's Resource Conservation and Recovery Act program, hazardous substances must be stored, handled and disposed of using measures that safeguard public health and the environment.
For more information on the Resource Conservation and Recovery Act, please visit the EPA's web site at: http://www.epa.gov/compliance/civil/rcra/index.html.
Search this collection of releases | or search all news releases
View selected historical press releases from 1970 to 1998 in the EPA History website.
Recent additions


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Liverpool star Suso fined for 'gay' tweet
Liverpool forward Suso has been fined £10,000 ($A15,000) after labelling teammate Jose Enrique "gay" on Twitter.
Suso sparked protests last month when he posted a photo on the micro-blogging website of Reds defender Enrique having his teeth whitened and added a tweet that read: "What fk is he doing? This guy is gay...he does everything except play football."
He later deleted the tweet and uploaded the photo with a different comment, saying: "I dunno what to say..."
But the Football Association, which earlier this year fined Ashley Cole £90,000 after the Chelsea and England star wrote a critical tweet about the governing body relating to the John Terry-Anton Ferdinand racism hearing, took exception to Suso's orginal comment and warned the 19-year-old about his future conduct.
The FA said in a statement: "Following an independent regulatory commission hearing yesterday (Monday, December 17 2012), Liverpool's Jesus Fernandez Saez (Suso) has been fined £10,000 and warned as to his future conduct for a comment posted on Twitter."
However, Enrique took to Twitter a few hours after the punishment was announced and criticised the FA for fining Suso over what was intended to be a good-natured exchange between friends.
"Is amazing how FA can fine my friend Suso Fernandez for a banter thing. Was just a joke!!!," the Spaniard wrote.
Suso had made a promising start to his Liverpool career after moving to Anfield from Cadiz in the close-season.
The Spain Under-21 international has made 13 appearances for Brendan Rodgers' side, including six starts in the Premier League.
Cole was punished after blasting the FA for casting doubt on his evidence at an FA hearing into claims that teammate Terry, whom Cole supported, racially abused Ferdinand - Terry was handed a four-match ban and fined £220,000.
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Shar Music in Home Trial
Our Safe, Easy, and Risk-Free In Home Trial Program allows you to try instruments or bows for a 7 day trial period.
Choosing Music
SHAR Guest Blogger, Kim Wren, talks about choosing the bass as a lifelong musical partner.
What to Look for in a Case
Our case purchasing guide takes you through the major features to consider.


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Overheard in a bar in British Columbia last week
"Any idea why 4 right feet have been washed up recently?"
"No, but I'd give my left foot to find out...."
Canadian police are probing a possible link between a left foot which washed up yesterday on a beach near Vancouver and four right feet which have since last year similarly made landfall on the coast of British Columbia, the BBC reports. The mysterious spate of seaborne foot landings began last August when two right feet came …
"Any idea why 4 right feet have been washed up recently?"
"No, but I'd give my left foot to find out...."
That's hilarious. What other jokes can we come up with? Let's see...
Canadian football team gaines 5 fold advantage?
I, for one, welcome our new podiatric overlords.
Ready to hand out to the inevitable "police think something is afoot" comment makers.
This will be an evolved sub-species of the Sole.
fathom it out when they get the 6th foot.
Coat, hat, door...
"We are certainly not discounting the possibility that this may be linked to the other recovered feet"
One of the other feet at most, surely.
"... why the coastline of British Columbia has grown five feet since last year"
Sea levels falling in this part of the world?
It's OK, I'm leaving.
Something is definitely afoot here.
it was reported in the BBC website...??
Easy crime to solve - all they need to do is ask around for any family that is missing a man with four feet on his left leg that went jogging a few months ago.
The show was televised on a local channel just before the feet started washing up.
Coincidence? I think not.
I couldn't believe the phrase:- While police say there is "no evidence that the feet were deliberately severed or removed by force",
Am I allowed to be a teenager and say WTF !
So feet are falling off now! This will be some Euro crap about feet being an imperial measure or something.
Mind you, at least I can tell my wife that my willy is now officially improved to a length of... (consults ruler) 15 or something. (For the ladies reading I'm being modest :-) or talking in light years maybe)
Anyway I'm going to get my 90cm coat now.
...While police say there is "no evidence that the feet were deliberately severed or removed by force"
You've got to love police understatement and caution when things like this happen. No evidence eh? Well, no, I suppose not. They probably just "fell off", eh?
...Daniel Day Lewis when you need him?
Someone had to say it.
Didn't they?
Welcome our moist plantigrade overlords.
Just a thought...
Or why do people throw their feet into the sea? And how can they walk away?
EAfH, puzzled
Top class gag there, Mr. Haines as always.
Does the shoe on the left foot match the shoe on any right foot?
That'll be the one that Daniel Day-Lewis has been looking for since '89...
"Evidence of deliberate removal" would be tool marks on the bones (saw or knife), or tendons ripped from attachment points.
My first question would be DNA analysis to see if there are any matches?
I feel morbid for even reading this article.
...I mean, come on, not one mention of the supposed owner species!!!
Bears have feet too you know, they just go by the more traditional name - Paws.
They are still feet though - I quote the always accurate (???) Wikipedia - http://en.wikipedia.org/wiki/Foot.
There are animals all over that page! ;-)
They don't have a leg to stand on.
They have been reporting on this news for a while here in the northwest. You would think they could give a little more detail, but they aren't. very interesting....
What's the probability that in a run of random foot tossings four right feet will turn up in a row?
Mine's the one that's been dragging in the mud on the right hand side.
that illegal imigrants have discovered a novel method of sneaking into Canada, having previously gained the assistance of Canadian beach combers via eMail message exchanges on Face Book or whatever .............
Can't find my coat since my last humiliating exit.
I am glad that these feet were wearing trainers as these are well known to provide excellent support and to enhance the biomechanical function of the foot - good choice. Professional opinion provided by my wife.
"We have no evidence that the feet were deliberately severed or removed by force" the detective was quoted as saying, "The shoelaces were still tied"
police think something is afoot... I'll take a large in black if you have it - I could do with a new coat.
So am I to understand that "Going clubbing in Canada" is to be replaced by "Going sole-searching in Canada"?
Maurice Shakeshaft: If they had another 18,224 they'd be in a completely different league.
I was watching a BBC Horizon TV program first aired March 2008 recently about the what had happened to the body of the late Alistair Cooke called "How Much Is Your Dead Body Worth ?" where the report detailed that an illicit trade in body parts from funeral homes is worth big dollars due to the lack of donors and the answer was a surprising USD$261,500-00 !
Now since one in seven literally die from assorted terminal cancers , these crooks motivated only by greed and lured by the high prices , they literally cut up the bodies in funeral homes to retrieve the wanted items charge the usual high fees and or merely hand wood ashes back to the grieving family and then live the high life on the proceeds from both jobs !
Hmmm , after viewing that program and seeing these reports , one can suspect that some one in Canada or the US just across the border have been dumping the unsold parts from supposedly cremated bodies from assorted funeral homes at sea but have literally failed to take into account the coastal currents and tide changes !
Truth can be stranger then fiction as some celebrities well know !
... will this mean a new series of Six Feet Under...?
are currently attempting to determine the whereabouts of Jake the Peg's children.
I reckon that sharks ate some bodies but couldn't digest the trainers which still had the foot inside!
If there are shark tooth marks on the trainers that might be a good clue...
Last time I looked, animals didn't wear trainers ... at least they don't here in the UK!
...it's just a legend (cough). Or at most a minor footnote in history. In fact, I'm surprised this story has legs, but if it is true, those responsible should tread carefully, since I'm sure the local plod will quickly step up to the mark.
Sounds like a good incentive to arrange in your will that the medical/scientific community can *buy* your body from your heirs for a "mere" $200,000 (saving them $61,500) and your family, instead of being $5,000+ poorer, will have a couple of hundred grand to play with.
Only people who will lose out are the unscrupulous bastards who'd charge your family > $5,000 and sell your corpse for $261,500.
BTW, I've also heard of the rogues stripping the coffins of all the fancy extras you bought to send off your beloved in style so they can sell them again to the next poor saps who want a fancy coffin with all the trimmings.
And do we really believe that that beautiful casket with all the brass on it that you *bought* actually goes into the furnace when the body is cremated?
Since "vulture" has already been appointed to journos, I propose "buzzard" to refer to funeral directors - they both live off the dead.
Oh, how clever.......
The story they've been floating around is that these are just the feet of regular old missing/drowned/dead bodies. The bodies sink once they've been in the water long enough, but the ankle joint is fairly weak and breaks as decomposition begins. Since the feet were all encased in trainers (which are buoyant), they float to the surface once free of the body. The foot washes ashore, leaving the rest of the body behind.
The fact that there have been four left feet is just coincidence.
Black helicopter because this is what they WANT you to believe.
...where people have attempted or even succeeded in committing suicide by leaping from bridges, cliffs etc it has been documented that from above a certain heights feet have a habit of snapping off on hitting the water.
Probably Gangster waste firms dumping hospital waste out at sea.
Also Christy brown had both feet, how ever his left foot was very dexterous and allowed him to paint and write.
My friend has 2 left feet....he wears flip flips.
I wonder what sneaker brand is so stinky that even sharks won't eat them?
"and your family, instead of being $5,000+ poorer, will have a couple of hundred grand to play with"
You don't seem to have considered the tax implications of the transaction. Death, taxes, coat, door, etc.
"You don't seem to have considered the tax implications of the transaction. Death, taxes, coat, door, etc."
Don't post anonymously - I like to know the names of those who brighten my morning with laughter.
So, I think it's moderately safe to assume that the victims were within 500 miles of the initial foot sightings?
Its stories like this that really creep me out as I don't really know if it's an actual tragedy or more of a foot fetish, gone slightly awry… I mean talk about the moral paradox (or in this case podidox)!
And not to discount any conclusive evidence that may suggest that sharks have a more discriminating palette or smart fashion sense to be seen consuming or caught dead wearing a single "trainer", let alone both, remains to be seen. I've personally seen distant fresh water relatives of sharks consume some things that best not be mentioned in a public forum, the best of which could hardly be more appetizing that a leather clad foot.
That's it, leaving without any fanfare as this is just wrong on so many different levels...
So if I need an organ I know were to go


text:
I have just set up a web site for my son's business. When viewing a page on the site a bar appears across the top with a bright yellow warning that reads:
"Unless you trust this site, avoid entering personal information or downloading anything from it." .... with a Check Point logo on the right.
How can I stop that from happening? This will be disconcerting to visitors to his site and counterproductive to having it.
He purchased the "Certified Domain" from godaddy.com and that logo and link is on his home page but it has no effect on the warning message.
Any help appreciated in removing this warning on his new site.


text:
Tests to clarify Rays prospect Lee's injury
By Bill Chastain and Adam Berry / MLB.com
ST. PETERSBURG -- Shortstop prospect Hak-Ju Lee flew down to the Rays' Minor League complex in Port Charlotte, Fla., on Monday to undergo a second MRI exam and determine a course of action for the torn ligaments in his left knee.
Mitch Lukevics, Tampa Bay's director of Minor League operations, said Lee will also visit Dr. Koco Eaton, the Rays' team physician. After Lee's MRI, the Rays should have a better idea regarding the extent of the 22-year-old shortstop's injuries, but it would appear likely that Lee will undergo season-ending surgery.
"There is a pretty good chance, I would say," Lukevics said. "Nothing's definitive, but there's a pretty good chance. ... With ligament damage, there usually is."
Lee, MLB.com's No. 56 prospect overall and the No. 5 prospect in the Rays organization, was involved in a rough collision at second base in the first half of Triple-A Durham's doubleheader on Saturday and left the field without putting any weight on his left leg. In addition to providing his usually excellent defensive work, Lee had gotten off to a much improved start at the plate, batting .422 with a 1.136 on-base plus slugging percentage through 15 games.
For now, Lee is doing limited work with the Rays' training staff in Port Charlotte, icing his knee and keeping it mobilized. Lukevics heard that Lee's spirits were "OK" on Monday but that the shortstop was unsurprisingly disappointed.
"Obviously, he's down in the dumps; it's only human nature to be down in the dumps," Lukevics said. "I know it's his goal -- I know it's our goal -- that we get him back. We're going to get him back playing at some point, and we're going to get a big leaguer out of this."
With Lee likely out of the picture for the rest of the season, former No. 1 overall Draft pick Tim Beckham should pick up most of Durham's starts at shortstop. The Bulls also have middle infielders Cole Figueroa and Mike Fontenot on their roster, however, so Durham can move those three all around the infield, much like the Rays do with their players.
"You know what we do -- we play them all all over, and it's a wonderful thing," Lukevics said. "It's all good. We're going to play them. ... We have to do these things in the Minor Leagues where, if a young man comes up here, hopefully it's not his first time playing at a position in the Major Leagues, the toughest level."
Free of pain, Scott anticipates brief rehab stint
ST. PETERSBURG -- Rays slugger Luke Scott will begin a rehab assignment with Class A Advanced Charlotte on Tuesday.
"I passed my test yesterday and came in today with no soreness, no stiffness," Scott said. "So I'm going to go down tomorrow to Port Charlotte, [Fla.], and start the process of coming back."
Scott has been out with a right calf strain since March 24. He does not believe he needs a long rehab stint.
"For me, all I have to do is get on the fastball," Scott said. "They want me to go down there and want me to play to see how my leg reacts. I've done this process before, and I know what I need to get better."
Scott said he won't be with the Rays by the time they go on the road following Wednesday night's series finale against the Yankees.
"Can't do that, because I'm going to play [on Tuesday], and Wednesday is going to be off to see how I react to [playing Tuesday]," Scott said. "And then Thursday, I'll play again and just go from there."
But Scott believes he'll be back soon.
"Oh, I'll catch them on the road trip at some point," Scott said.
Whether Scott will play the field during his rehab stint remains to be seen. What's known is that he will be the Stone Crabs' designated hitter on Tuesday, and he expects to have four at-bats.
Instincts behind Maddon's advice to Yunel
ST. PETERSBURG -- Yunel Escobar went 3-for-4 with a home run and a double in Sunday's 8-1 Rays win over the A's. After spending the previous five games hitting ninth for the first time since his rookie season, Escobar moved up to sixth in the order on Monday night against the Yankees.
It didn't take long for the move to pay off, as Escobar took ace left-hander CC Sabathia deep with two outs and a runner on in the bottom of the first, extending the Rays' lead to 4-0. Escobar, who was hitting .089 (4-for-45) over his first 13 games of the season, credited Joe Maddon for the turnaround, as the Rays manager gave him a tip.
Maddon seemed surprised Escobar had mentioned that he had given him a tip, then the manager explained the advice he offered his shortstop.
"I'm an old hitting coach by trade," Maddon said. "There are different things I used to do back in the day. The part of him that I had seen or thought I had seen, there were a lot of balls off the end of the bat, and he's really a strong fellow. It's not like he's late; he's never been late. He's not long, he's not late, he's on the end of the bat -- so what's going on here?
"I just suggested a bigger, longer, heavier bat, and just thinking the logic through that. He just has a little more coverage with the end of the bat and it's a little bit heavier, which forces you to use your hands more and not so much your arms. That was pretty much it. And he's responded pretty well."
Rehabbing Oviedo sticks with Rays at home
ST. PETERSBURG -- Right-handed reliever Juan Oviedo has been in uniform and in the Rays' dugout during the club's current homestand.
"We wanted to keep him attached to the team somehow," Rays manager Joe Maddon said, "and he pointed out to me yesterday that he is 3-0. So you're always welcome here. But that was part of the plan during Spring Training. [Head athletic trainer] Ronnie [Porterfield] and I talked about it in camp, that when we're at home and we can have him here, we should, just to let him become absorbed into the group."
Known as Leo Nunez during his days as the Marlins' closer, Oviedo logged 92 saves from 2009-11 -- eighth most in the Major Leagues in that span -- but he had Tommy John surgery last Sept. 6 and is still in the midst of his recovery.
Maddon said he did not know if Oviedo would be fully recovered in time to pitch for the Rays this season, though he allowed, "I did hear something that the end of the year was possible."
Maddon added: "This guy is going to be a big part of our future."
Bill Chastain is a reporter for MLB.com. Adam Berry is a reporter for MLB.com. Follow him on Twitter at @adamdberry. This story was not subject to the approval of Major League Baseball or its clubs.


text:
emoze pushes email, calendars, tasks, and contacts to mobile devices as soon as they are sent, delivering real-time synchronization. emoze software is simple to download, register, and use. All emoze users need a data package from a mobile service provider. emoze works across any mobile service provider network and has users in almost every country around the globe. emoze delivers data to and from all leading brands of mobile device. It supports all popular email data sources and even social community messaging such as Facebook, and all popular mobile device operating systems. The emoze service is independent of network technology and works on any wireless GSM, CDMA, or Wi-Fi network including 2.5G and 3G.
W-Meter is a tool for testing the conformance and evaluating the performance of 802.11 products. The tool is meant for injecting arbitrary frames to test the conformance of any wifi product. The tool has templates for generating various kinds of frames which can be customized for generation of specific frames. The tool can be used to elicit responses from other wifi cards to check the conformance of the card to the protocol. At present, 802.11g frames are completed.
Bluelog is a Bluetooth site survey tool, designed to tell you how many discoverable devices there are in an area as quickly as possible. Bluelog differs from most Bluetooth scanners in that it prioritizes speed of reporting over anything else (i.e. it doesn't spend time trying to pull detailed data from a device) and doesn't require any user intervention to function. As the name implies, its primary function is to log discovered devices to file rather than to be used interactively. Bluelog could run on a system unattended for long periods of time to collect data. In addition to basic scanning, Bluelog also has a unique feature called "Bluelog Live", which puts results in a constantly updating Web page which you can serve with your HTTP daemon of choice.
WiFiDB is a set of scripts that is intended to manage wireless access points found with the Vistumbler wireless scanning software. WiFiDB also has support for importing Netsumlber summary files and Wardrive for Android database files. WiFiDB can also export access points with GPS to a Google Earth KML file, so you can see where the access points are located.
NetSpot is a simple and accessible wireless survey tool. It allows you to collect, visualize, and analyze Wi-Fi data using any MacBook. NetSpot can visualize your wireless network by building a visual Wi-Fi map. It will let you see dead zones without coverage and place hotspots (access points) correctly. Using the visualized map of collected wireless data, you will also see channels' load and will be able to decide how to optimize your Wi-Fi site better. NetSpot is a powerful analyzer of Wi-Fi coverage. It will gather all the information needed for analyzing radio signal leakages, for discovering noise sources, for locating effective access points, etc. You can also use NetSpot as a powerful wireless network planning tool.
OsmoSGSN (Serving GPRS Support Node) is the central core network node of a GPRS/EDGE/UMTS cellular network. It is an experimental SGSN implementation, exposing a Gb interface (NS/BSSGP) towards the BSS and the GTP protocol towards the GGSN. It is not yet ready for production use, but primarily for laboratory and research use.
SIMtrace sniffs communication between a mobile phone and a SIM/USIM card. This can be useful for research and analysis, and for developing mobile phone and SIM toolkit (STK) software. The SIMtrace hardware is also capable of MITM (man in the middle) attacks on the SIM-MS communication.


text:
Oblique strain could sideline Gardner rest of season
By Bryan Hoch / MLB.com
BOSTON -- The Yankees are preparing to play through the remainder of the regular season without Brett Gardner, who suffered a Grade 1 strain of his left oblique on a checked swing during Thursday's 6-5 win over the Orioles.
Gardner had an MRI performed on Friday in New York that revealed the strain. Losing the 30-year-old, who performed well as the club's leadoff hitter and everyday center fielder, will be a tough blow as the Yankees attempt to chase a playoff spot.
"It's a big loss," manager Joe Girardi said. "We've had to overcome a lot over the course of this year and we're going to have to continue to do that. People are going to have to step up in his absence."
Girardi said that it is possible that Gardner could return first as a pinch-runner, then as a full player, if the oblique strain heals quickly. However, Girardi also acknowledged that it is possible Gardner has played his last game of the regular season.
Gardner had been one of the Yankees' few constants in an injury-riddled season, batting .273 in 145 games while setting career highs with 147 hits, 33 doubles and eight home runs.
"He's been really good for us -- offensively, defensively, getting us off to quick starts," Girardi said. "And he's going to be out a while."
Curtis Granderson was in the lineup batting leadoff and playing center field on Friday as the Yankees opened a three-game weekend series with the Red Sox.
"It always helps when you have players who can fill in," Girardi said. "But [Gardner is] an element that, we talked about last year how we missed that element a lot. And guys are going to have to step up. There's no doubt about it."
Sore hamstring to keep A-Rod from playing third base
BOSTON -- Alex Rodriguez's speed had already moved into the slow lane following two hip surgeries, but a tight left hamstring now has him putting the caution flashers on.
Rodriguez did not move well on the basepaths in the seventh inning of Friday's 8-4 Yankees loss to the Red Sox, and Rodriguez said that he will be unable to play third base until at least next week because of concern that he will further injure the leg.
"I'm just trying to go where it doesn't blow out. That's pretty much it," said Rodriguez, who has been the Yankees' designated hitter in three straight games.
Rodriguez said that he was feeling some improvement in the hamstring, which he said knotted up on Tuesday in Baltimore, but manager Joe Girardi noticed Rodriguez laboring as he moved to third base on Robinson Cano's two-run double in the seventh.
Girardi had planned to play Rodriguez at third base on Saturday, but he said that his mind had changed after watching Rodriguez run the bases. Rodriguez said that he did not think he could play third base until at least Tuesday in Toronto.
"I'm not sure. I'm coming in every day and doing treatment with [head athletic trainer] Stevie [Donohue]," Rodriguez said. "Hopefully sooner rather than later. But I know that Monday day off will be key for me and hopefully maybe Tuesday."
Girardi said that he believes Rodriguez, who was 0-for-3 with a walk on Friday, will still be available as a DH on Saturday.
"I'm not sure what I'll have [Saturday]," Girardi said. "I'll have to check with him. It's a concern of mine."
Granderson prepared to take over center field duties
BOSTON -- Curtis Granderson knew it was not good news when he was told to grab his glove and head to center field, replacing Brett Gardner for the bottom of the first inning on Thursday.
Now that an MRI has confirmed that Gardner has a Grade 1 strain of his left oblique and may miss the remainder of the regular season, Granderson said that he is prepared to fill in as the Yankees' everyday center fielder, as well as in the leadoff spot if needed.
"Anytime you lose anybody, it's always a tough loss, no matter who it happens to be in this organization," Granderson said. "You don't want to see anybody get hurt. Gardy has been playing well for us all season long, and he's done some amazing things both offensively and defensively.
"To have him go down, the way he is and to not be sure exactly when he might come back, that's definitely a tough thing to handle. At the same time, we've got to continue to move on."
Granderson has been limited to just 46 games himself entering play on Friday, suffering a fractured right wrist in his first Spring Training game and then breaking his left hand in May, both on hit-by-pitches.
Granderson has bounced around the lineup, hitting in all of the first seven slots, and he has also played all three outfield positions. Granderson said that moving around to a variety of roles has not bothered him, because he is just happy to be playing.
"It's fun. This is great," Granderson said. "We're in the thick of things, ready to move forward and have a chance to get to the playoffs. It has been fun, it's been unscripted to a tee. Anything that you could think of happening has happened, and I'm sure there's a lot more that will happen that is unpredictable. That makes it fun."
Bombers bits
• Catcher Austin Romine (concussion) took some swings on Friday and said that he is continuing to feel better. Romine took an ImPACT test on Thursday, and Girardi said that although it went well, it is possible Romine won't be available to play this weekend.
• Left-hander Boone Logan (left elbow tightness) told Girardi that he was feeling better, and Girardi said that he is "hoping that he turned the corner." Logan shut down a throwing session on Thursday in Baltimore after just five tosses due to discomfort.
• On this date in 2011, Mariano Rivera recorded his 600th career save in a victory over the Mariners at Safeco Field. The final out was recorded when catcher Russell Martin threw out Ichiro Suzuki attempting to steal second base.
Bryan Hoch is a reporter for MLB.com. Follow him on Twitter @bryanhoch and read his MLBlog, Bombers Beat. This story was not subject to the approval of Major League Baseball or its clubs.


text:
I thought I better pipe up - yep, I'm from the Great White North (Bob and Doug Mackenzie anyone?).
I'm in Ontario (I won't get much more specific in a public conference, sorry!)
The Lex is marvelous because when I was at the Midnight Release party for Breaking Dawn at our local Chapters, I wore my Lexicon shirt. I was feeling slightly vulnerable (when you are a 30-something year old fan surrounded by teens at an event, you can feel a bit alone in a crowd) when someone came up to me and gave me a high-five because she said "The Lex? I love that place!" She was wearing a cool Volturi outfit for the costume competition. If she's on here, I want to let her know that she made my night (okay, getting Breaking Dawn was the highlight, but she was second place!)
Sadly, I discovered Twilight two weeks after the big visit by Stephenie to Canada. Just my bad luck! Maybe one day ...


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ASSATEAGUE – Assateague Island National Seashore will get a fiscal shot in the arm with the announcement last week of over $900,000 in American Recovery and Reinvestment Act funding for a handful of projects.
Last week, the National Park Service announced nearly 800 projects totaling around $750 million will be completed with federal economic stimulus funds with the majority of the identified projects in the northeast region from Maine to Maryland, which will receive roughly $6.6 million for 27 identified priority projects in the state.
Included on Maryland's list of projects is a combined $908,000 for two projects in the Sinepuxent District of the Assateague Island National Seashore. The list includes $460,000 for the installation of fire suppression systems at Assateague Island National Seashore and another $448,000 to rehabilitate all of the boardwalk areas throughout the Sinepuxent District on the island.
All of the projects on the list, including the two on Assateague, are long-standing priorities of the National Park Service and meet the criteria carefully spelled out in the Recovery Act. For example, they were all chosen because they address the Department of the Interior's highest priority mission needs, generate the largest number of jobs in a short period of time and create lasting value for the nation's park-visiting public.
Assateague Island National Seashore Superintendent Trish Kicklighter said this week the projects identified on the barrier island meet several of the criteria spelled out in the Recovery and Reinvestment Act.
"At Assateague, the funding will be used to improve the visitor experience while addressing safety issues in some of the facilities," she said.
U.S. Department of the Interior Secretary Ken Salazar stressed the importance of utilizing the federal funding to shore up the national parks during a time when they need it the most. The projects identified on the list will also provide an associated economic boost in the communities where they have been identified.
"These projects are an investment in America's future that will create jobs, stimulate the economies of the local communities and get our country moving again," he said. "They are also an investment in telling the story of America to future generations through our national parks by conserving our awe-inspiring landscapes, our rich culture and our great heritage."
Besides Assateague, other significant projects are listed for Antietam, Fort McHenry, the C&O Canal and Catoctin.


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[SMH | Text-only index]
Date: December 28 2012
Western Sydney coach Tony Popovic will have a welcome headache at the selection table when he decides on his starting line-up for Tuesday night's A-League clash with Melbourne Victory at Parramatta Stadium.
Popovic's decision to make six changes to his starting line-up against Perth Glory on Thursday night paid handsome dividends, with the Wanderers holding on for a vital 1-1 draw away from home.
Star Japanese recruit Shinji Ono, Youssouf Hersi and German defender Jerome Polenz were rested altogether, while in-form striker Mark Bridge and Dino Kresinger started on the bench.
Suspended midfielder Adam D'Apuzzo rounded out the mass changes.
The Wanderers were on track to snare all three points following Labinot Haliti's beautiful curling effort in the 38th minute, but Glory substitute Nick Ward thundered home a powerful volley in the 87th minute to ensure a share of the spoils.
The result kept Western Sydney firmly entrenched in the top-four, but their title credentials will be put to the test over the next nine days with games against Victory and the ladder-leading Mariners.
Popovic was proud of the effort his charges displayed against the Glory, and said he was yet to decide who to bring back into the starting line-up against the Victory.
And the former Socceroos defender said his decision to rest so many players - less than a week after the club's 6-1 demolition of Adelaide - was a necessary one given the searing Perth heat and the short break between games.
"We've just had our third consecutive six-day turnaround, and we need all our players as fresh as possible," Popovic said.
"The players that played today, they deserved an opportunity as well."
The Wanderers have now snared three wins and a draw from their past four games, but Popovic said it was too early to get carried away, especially with games against the Victory and Mariners to come.
"I'm not sure where we stand in the title race. For us they're two games we're looking forward to," he said.
"We're not halfway through the season yet. There's a long way to go. We'll be judged after 27 games. That's when your ladder position is important."
This material is subject to copyright and any unauthorised use, copying or mirroring is prohibited.
[ SMH | Text-only index]


text:
Because — for the humor-impaired — there's been way too much anger, bitterness, feuding and rancor over this subject (a subject which, among sensible adults, there should really be not a whiff of controversy at all) that I think a little calming levity is overdue.
(For those curious, this comes from the amazing Tumblr feed Black and WTF, and sports this caption: February 27, 1923. "Miss Alice Reighly, 1409 Harvard Street, president of Anti-Flirt Club, which has just been organized in Washington, D.C., and will launch an 'Anti-Flirt Week' beginning March 4. The club is composed of young women and girls who have been embarrassed by men in automobiles and on street corners." So, you know, it's not a new problem.)