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Search results
Registration now open for November!
". https://epn.osu.edu/events/epn-webinar-november-10-2020-cooperating-sustainable-development_blank Marquee Slide Gray Marquee ... Registration is now open for our November EPN Webinar virtual program, "Cooperating for ... Sustainable Development ...
What Does it Take to Get Students Learning Outside?
cooperation leading to greater achievement. Emotional Development: Hands-on outdoor programs appear to have ... management techniques work? The answer to all these questions can be found in developing one's own capacity ... communication skills and curiosity to develop a high-quality experience for students. Why would an educator ...
Best Practices for Effective and Efficient Pesticide Application
Specialist, Department of Food, Agricultural and Biological Engineering, The Ohio State University Applying ... problem. These major principles include: Positive identification of the pest(s) Selecting the pesticide ... Biological Engineers) developed a standard used by nozzle manufacturers to classify nozzles based on droplet ...
Creating, Preserving, and Reengaging the Paris Agreement on Climate Change
targets. If President Trump wins, we will look at what the future holds for global climate cooperation in ...
Farmer and Farmland Owner Income Tax Webinar
eventual payments Qualified Business Income Deduction, sales to cooperatives, lease income Other topics The ...
Ohio Certified Crop Adviser Pre-Exam Training
Nutrien and past OSU Soil Fertility Specialist; Greg LaBarge, CPAg/CCA – Professor OSU Extension and Field ... Specialist Agronomic Systems; Bruce Clevenger, CCA – Associate Professor OSU Extension, Area Leader and ... County AgNR Educator; Harold Watters, CPAg/CCA – Associate Professor OSU Extension and Field Specialist ...
Farming and parenting—a tough juggling act
This news article was originally published on the website of the College of Food, Agricultural, ... the news ...
Ohio State students' sustainability behavior and knowledge continues to grow, according to an annual survey
Development. Read more here. Questions about the survey? Contact the ESS Lab via email at: firstname.lastname@example.com ...
Hamilton County 4-H Update 10-7-2020
Development is moving forward with five projects to improve the program and involve more youth! 1.) ... continue to develop a strategic plan for Hamilton County 4-H Youth Development. 2.) New Area Leader Pat ... previous design. 5.) In Person Programming Hamilton County has moved back into a Level 3 (Red) emergency. ...
Extended Drydown in Corn
how the weather conditions impacted corn growth and development this year. In many parts of Ohio in ... following planting may have also contributed to poorer root development in 2020. Poor root development may ... corn crop may have experienced a sort of "delayed development" this year. Temperatures were below the ...


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Software Development
Website Designing
Mobile App Development
Search Engine Optimization
Software Company in Jashpur
Parakite Solutions is an Offshore Software Company In Jashpur. We have more than 5 years of experience in Developing Software's and Web Portals. With Our Software Applications We are able to satisfy our customers need and requirements. We have a list of our clients portfolios through which customers can trust us. Our Company Provides Services Offline & Online in Jashpur Also.
Client's Satisfaction is our topmost priority So we invest a lot of time in understanding clients requirements. We do a lot of research and competitor analysis before we begin to develop software. Thats why we are awarded as most creative creative Software Company In Jashpur
Our Services Includes Software Development, Website Designing, Mobile App Development, Website Maintenance and many more. We can Provide Services Online and Offline Both. Our Expertize Products are School Software, Institute Software, Online Exam Management System, Nidhi Society Software, Credit Cooperative Society Software, GST Billing Software and many more.
1. We are the largest software development company in Jashpur
2. We have 5+ of experience serving clients in Jashpur area and surroundings. So we are here to stay and deliver the best Software.
3. We are the only Custom software development Software company in Jashpur. We can tailor the software as per need of our clients.
4. Our highly Skilled and Experienced team will do the software development process very swiftly.
5. We use all latest technologies with the best practices to develop Software.
6. Our customer service the best in the market not only in Jashpur but in whole India.
We are one of the Top software companies in Jashpur that are strategic in approach and we ensure that we give the complete Strategy of the product before commencing the development work on a particular project. We ensure that both the frontend and backend team work in sync to get the best results.

subset: commoncrawl


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Search Constraints
You searched for: Author Geological Survey (U.S.) Remove constraint Author: Geological Survey (U.S.) Subject Topographic maps Remove constraint Subject: Topographic maps Subject Digital elevation models Remove constraint Subject: Digital elevation models Place Arkansas Remove constraint Place: Arkansas Place Connecticut Remove constraint Place: Connecticut Place Arizona Remove constraint Place: Arizona Year 2012 Remove constraint Year: 2012
Search Results
1. 100-Meter Resolution Color-Sliced Elevation of the Conterminous United States
2012. Geological Survey (U.S.). This layer is a georeferenced raster image displaying color-sliced elevation data for the Conterminous United States at 100-meter resolution, using... National Atlas of the United States.


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The smell of gun smoke. The sounds ringing in your ears.
Size: 24x16
Paper: We use Metallic paper for our posters. Metallic paper features a unique pearlescent surface that offers sharp details and beautifully saturated colors. Plus, metallic is strong - resistant to tearing and curling without any additional lamination. This paper uses a patented combination of film and laminate layers that result in striking, three-dimensional, lasting images on an ultra-bright background.


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Our selection of new mom & baby items are our way of saying congratulations! Being a new mother is a feeling unlike any other. It's also hard work. Our selection of new mom &baby items will help make all of the hard work a little easier. We have a full line of promotional products designed to help out new moms and babies everywhere.
We want to help new mothers anyway we can. For starters, we provide a hand book especially designed to assist a new mom & baby. This handbook is under 2 dollars and provides some critical mommy information. We also have little pamphlets that will cover the nutritional needs of newborns. With our line of new mom & baby products, moms everywhere can ensure that their child is receiving the proper nutrition. Choosing the proper nutrition early on can help fight against childhood and lifelong obesity.
We have more than educational messages in our selection of new mom & baby products. We have great baby t-shirts. These shirts are sure to bring a smile to the faces of anyone looking at your new baby. For instance, our mom's café open all day t-shirt is very popular among newborns with a sense of humor. We hope you enjoy our selection of new mom & baby and would once again like to congratulate the new mothers out there.


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In accordance with an International Agreement between the European Union and the United States, Brussels Airlines transfers to the US Department of Homeland Security (DHS) certain travel and reservation data (PNR) about passengers flying between the European Union and the United States.
US authorities use this information for preventing and combating terrorism and other transnational serious crimes. These and other data may also be used for checking against lists of passengers raising aviation security concerns.
The information will be retained for fifteen years and may be shared with other authorities. Information related to a specific case or investigation can be kept longer until the case or investigation is archived.
You can get more information on request or see the Frequently Asked Questions (FAQ):
United States law requires airlines operating flights to, from, or through the United States (US) to provide the US Department of Homeland Security (DHS) with certain passenger data to facilitate safe travel and to guarantee US security by carrying out advance risk assessments of passengers. The passenger data falls into two categories.
These FAQs relate primarily to PNR data as this is regulated in accordance with the International Agreement signed on 26 July 2007 between the European Union and the United States. The European Union will make sure that air carriers comply with these obligations. Brussels Airlines has to comply with these requirements.
For a more detailed explanation of the way DHS handles PNR collected from flights between the European Union (EU) and the US, please refer to the international agreement and the accompanying letter of DHS, which are published in the Official Journal of the European Communities L 204 of 4 August 2007, available here click here.
DHS uses passenger name records (PNR) for flights between the EU and the US for the purposes of preventing and combating:
PNR may be used where necessary for the protection of the vital interests of an individual, or in any criminal judicial proceedings, or as otherwise required by law.
By legal statute (title 49, United States Code, section 44909(c)(3)) and its implementing (interim) regulations (title 19, Code of Federal Regulations, section 122.49b), each air carrier operating passenger flights in foreign air transportation to or from the US must provide DHS with PNR data collected and contained in the air carrier's reservation and/or departure control systems.
DHS explained in a letter of 26 July 2007 how it handles the collection, use and storage of PNR.
These conditions concern, in particular:
On the basis of these assurances by DHS, the European Union and the United States concluded an international agreement signed in July 2007, and the European Union will now make sure that air carriers make available PNR data as required by DHS.
Certain PNR data identified as "sensitive" may be included in the PNR when it is transferred to DHS. Such sensitive PNR data would include certain information revealing the passenger's racial or ethnic origin, political opinion, religion, health status or sexual preference. DHS has undertaken that it will not use any sensitive PNR data that it receives and has put in place an automated filtering programme so that sensitive PNR data is not used in principle.
However, sensitive PNR data may be used in exceptional cases, such as where the life of an individual could be imperilled or is in serious danger.
DHS may share PNR data with other US government authorities that have counterterrorism, law enforcement or public security functions, for the purposes of preventing and combating terrorism, transnational crime and public security (including threats, flights, individuals and routes of concern).
DHS may share PNR data with government authorities in third countries, but only after they have considered the intended use of the information and the ability to protect it. Unless it is an emergency, any exchanges of data require data protection measures to be incorporated that are comparable to those set out in the international agreement between the EU and the US.
DHS will keep PNR data for fifteen years. Information related to a specific case or investigation can be kept longer until the case or investigation is archived.
DHS will keep a log of access to any sensitive PNR data and will delete the data within 30 days once the purpose for which it has been accessed is accomplished, and it is not required by law to keep it for longer. DHS will provide notice, normally within 48 hours, to the European Commission (DG JLS) that sensitive data has been accessed.
Any passenger, regardless of their nationality or country of residence, may request information about their PNR and have inaccuracies corrected.
You can find information on DHS policies on access to records and personal information at http://www.dhs.gov/xfoia/editorial_0579.shtm. For more information, please contact the DHS Privacy Office's FOIA Program:
The Privacy Office
U.S. Department of Homeland Security
245 Murray Drive SW
Washington, DC 20528-0550
Toll-free: +1-866-431-0486
Phone: +1-703-235-0790
Fax: +1-703-235-0443
Email: firstname.lastname@example.com
DHS policies on redress can be found at www.dhs.gov/trip.
If you need any help with making your request, you can contact your national data protection authority. For contact details of the DPA in your country, please click here.
You can get more information about how your personal data is handled by contacting our Service Centre.


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Ausfish Fishing AustraliabyPublished on 17-09-2015 03:13 AM
Animated Fishing Knots easy to follow animated fishing knots. How to throw a Cast Net online videos of two ways you can throw a cast net. How to fillet a Flathead online video of how to fillet a flathead. How to to fillet a Snapper online video of how to fillet a snapper How to de-vien a raw prawn online video of how to de-vien a raw prawn Fishing with Fitzy Freshwater location maps, articles and information. Becoming A Better Fisherman Guide on how to catch fish in Australia Queensland Transport Maritime division Fishing regulations In Queensland Qld DPI Fishweb, Minimum and Maximum sizes and bag limits, rules, etc.byPublished on 17-09-2015 03:43 AM
The following information is provided courtesy of ALVEY REELS
The sole purpose of this information is to help you become a better angler - to give you good basic information that will make your fishing trips in Australia more enjoyable and let you fully enjoy this relaxing, fulfilling sport.
Alvey Reels Australia, Snyder Glas Rods and Platypus Lines are all privately owned Australian companies that manufacture in Australia. The following information draws from the 200 years of combined knowledge gained from manufacturing a wide range of quality products to suit Australian fishing conditions and from top competitive anglers.
Many books have been written giving instructions to the novice or beginner in the art of angling. Most have defeated their purpose by showing every conceivable knot, serving only to confuse and discourage the reader.
This information shows comparatively simple methods which have been developed and accepted by top competitive fisherman to give the maximum efficiency. Having been proved conclusively in keen competition, these methods can be recommended with confidence as being worthy of a try by all fishermen.
Important Environmental Notice
Remember for fishing to improve we all have to be responsible ... don't litter empty bait bags, beer ring packs ... discarded fishing line should never be thrown in the water ... always return undersized fish, never take more fish than you need.
Fishing Styles
Fishing VarietiesbyPublished on 17-09-2015 03:40 AM
Note, cast nets are illegal in some states, check your local laws.
How to Thow a Cast Net - demonstrated by Steve Brown Media Player PDA iPod
How to Thow a Cast Net - demonstrated by Dave "Nugget" Downie Media Player PDA iPodbyPublished on 17-09-2015 04:36 AM
- This allows you to post commercial ads in the classifieds section
- Your ads are also displayed in your signature on all your forum posts
- You can have a commercial image and link in your signature that is displayed on all of your forum posts
- You can post commercial posts on the forums
- Your profile will clearly show that you are an Ausfish Advertiser so members will know to support you as you support them.
- Your website and contact information can be listed in your member profile
You can now add your business to our trades, services and products directory. This is an online directory that you can add your business to and have complete access to change and update your own information.
Click here to try it FREE for 120 days


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Whole Australian biodynamically grown raw almond kernels.
Gluten Free, Dairy FreeWheat Free, Vegetarian, Vegan, Paleo
This product has been packed inside our facility which handles products containing wheat, soy, peanuts, sesame seeds, tree nuts, eggs and dairy. While we do our best to avoid cross contamination, we cannot guarantee this. We recommend people with severe allergies or adverse reactions to these ingredients avoid this product.
We will create your wholesale account send you the details.
You can start ordering and you'll have organics delivered to you in no time.
2 to 3 business days is our standard delivery time. This also depends on your order details.
We deliver Australia-wide. In Melbourne, deliveries are done by our team using our vehicles.
Yes, our team will confirm the time and order details with you before pick up.
We have no minimum order size and we can handle orders up to 500 kilograms.
If a product you want is out of stock, let us know and we will tell you when we have more.


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Georgia Rose Iponiki
- 34,07€ 140,99€
- Référence : t1d2sQlg
- Disponibilité : Disponible
Options disponibles
The French brand Georgia Rose is a fashionista's dream. With a virtually infinite range of beautiful shoes and accessories to meet all your fashion requirements, you'll find it hard to resist this unique brand! From city basics that are functional and easy to wear, to glamourous shoes for special moments and weddings, Georgia Rose has a shoe for every occasion. The Georgia Rose woman is multi-faceted - dynamic, feminine, dreamy and glamourous. She is interested in contemporary fashion without being a slave to it, and likes to stand out in a crowd with her individual style. Georgia Rose understands that a woman's life is made from a million moments, and her shoes reflect this. Georgia Rose is becoming our new best friend with her styles and designs that are excellent value for money, and that can all be accessorized with the Georgia Rose range of bags to compliment your shoes. With a wide range of sizes (many styles are available from a UK 2 to a UK 10), at purse friendly prices, treat yourself with Georgia Rose, and embrace her happy go-lucky spirit and on-trend designs into your life!
Georgia Rose Iponiki


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Mini Medallion Bracelet
Our mini medallion, worn on a delicate chain that's perfect for layering, is a mighty symbol of strength, hope, and resilience.
- 10mm medallion
- 14k yellow gold vermeil (thick 14k yellow gold coat on solid sterling silver base)
- Awe hangtag with peridot, the stone of renewal and self-empowerment
What shipping options are available?
Awe is pleased to offer complimentary shipping and returns on all orders $100 or more, shipped within the United States. All of our products are shipped via USPS, Signature Required. Please make the necessary arrangements to have your package signed for. Domestic orders should arrive within 4 to 5 business days. Domestic two-day and overnight shipping are also available via UPS, with additional fees applied at checkout. Please allow 5-7 business days for your order to be fulfilled.
Can I ship my order to an International location?
Yes!Orders over $200 USD receive free shipping to Canada and orders under $200 USD to Canada will be charged a flat rate of $15 USD shipped via UPS or DHL. We ship to any country in the world via USPS, DHL or UPS for a flat rate of $35 USD. ALL INTERNATIONAL ORDERS ARE FINAL SALE. Please note that Awe Inspired is not responsible for paying additional VAT charges for customs clearance and will not lower the value of orders to avoid potential customs fees. All customs fees are separate from the shipping cost and are the sole responsibility of the customer. Any costs, duties, fees or taxes cannot be refunded.
How can I follow the status of my order?
Once your order has shipped, you will receive an email with a USPS or UPS tracking number.
What is your return policy?
We want you to be completely in AWE of your purchase. If for any reason you are not satisfied, we will gladly accept items for full refund, exchange or merchandise credit. The item must be returned in new, unworn condition within 30 days, and accompanied by your order number. NOTE: Full refunds are not guaranteed and subject to our review. Engraved orders are non-returnable. Please allow up to 10 business days once your return has been received for your credit to appear on your statement. International orders are final sale.
How can I make returns or exchanges?
For returns or exchanges, please email firstname.lastname@example.com with your order number and we will email you a printable USPS shipping label and instructions. Please note that returned or exchanged merchandise should include all AWE packaging. ALL INTERNATIONAL SALES ARE FINAL.
Can I exchange an item I just received for a different item on your site?
We will gladly accept items for exchange, so long as they are returned unworn within 30 days of purchase. Please note that all international sales are final and cannot be returned or exchanged. Engraved or Special/Custom orders are non-returnable. Please allow 10 business days for your exchange to be processed.
How long does it take to process returns?
Please allow 10 business days for returns and exchanges to be processed. Please note that all international sales are final and cannot be returned or exchanged.
Our diamonds are sourced through suppliers that adhere to the Kimberley Process and the World Diamond Council's System of Warranties, preventing the distribution of conflict diamonds.
Core to our company's values is paying equal and fair wages for women and men, providing proper health care for all those involved in the AWE product cycle, and treating our environment with care. Our manufacturing partners meet all Department of Environmental Protection (DEP) certifications pertaining to employee health as well as environmental consciousness during all manufacturing and disposing of waste. Our manufactures also comply with the Fair Labor Association's (FLA) workers protection standards to ensure a safe environment for all employees.
You may also like
- 10mm medallion
- 14k yellow gold vermeil (thick 14k yellow gold coat on solid sterling silver base)
- Awe hangtag with peridot, the stone of renewal and self-empowerment
Ethically Sourced &
Made to Last a Lifetime
Free Shipping & Returns
on All U.S. Orders
20% of Every Purchase
is Donated to Charity
Honest Pricing
Every Time


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RGB & Multi-Color LED Strip Light
Our RGB and multi-color LED strip light allows customers to have full control over the color of their lighting. With three or more LEDs on each node, users can seamlessly blend colors to achieve the perfect hue. Color-changing LED lighting has the power to completely transform a lifeless room into an inspiring statement. Use your imagination and choose your desired color to set the mood.
5-Year Warranty
Quality matters and we stand behind our standard strip lights with a 5-year warranty. We know that your projects have to perform flawlessly and failures are not an option. You can rest assured knowing you are getting high-quality strip light that you can depend on.
Beyond RGB
Mix RGB with the addition of a 4th, 5th or 6th color to create bolder and more vibrant colors. These limitless color options are perfectly suited for the creative challenges of scenic, stage and retail environments.
UL Listed
Virtually all our strip lighting is UL Listed in the low-voltage luminare category so you can get the approvals you need.
Engineering is the lifeblood of Environmental Lights and we work tirelessly to ensure we create dependable lighting systems that scale to your needs. Our team is on call to make sure everything goes right. If you have any questions, please call 888-880-1880 to get detailed product information from highly-trained technical specialists.
MaxRun RGB 4040 LED Strip Light - 84/m - Sample Kit
Product No:
RGB-CC4040MR-84-10-kitPrice Per Kit : $26.80
MaxRun RGB 4040 LED Strip Light - 84/m - 10m Reel
Product No:
RGB-CC4040MR-84-10-10mPrice Per Reel : $336.25


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- 1 Protocols
- 1.1 Cell Culture Protocols
- 1.2 Protein Protocols
- 1.3 DNA Protocols
Cell Culture Protocols
Bacterial Cell Cloning / Inoculation of Cells
Entire procedure is done under flame
- For small test tubes add:
- 5mL LB media
- 500µL 20% glucose (final conc. 0.2%)
- 7.35µL of CM (final conc. 50µg/mL, working stock: 34 mg/mL)
- Add the cells
- If from glycerol stock, stab with pipette tip.
- If from LB-agar plate, pick the colony (mark it on the cap) and stab the colony
- Incubate on shaker overnight
Bacterial Cell Culture Solutions
LB media
- For 1L of LB media add:
- 10g Tryptone
- 10g NaCl
- 5g Yeast extract
- Fill to 1L with DI water
- Autoclave at P6 setting
LB-Agar Plate media
- For 1L of LB-Agar media, mix:
- 960mL water (860 mL if adding glucose)
- 10g Tryptone
- 10g NaCl
- 5g Yeast extract
- 15g Agar (add agar last)
- Autoclave (liquid option): 30 min. sterilization / 20 min drying
- Cool to 50˚C in water bath
- Next three steps are under flame
- (if adding glucose: add 100mL of 20% glucose, final conc. 2% glucose)
- Add antibiotics to the specified final conc:
- Chloramphenicol (CM): 50 µg/mL
- Ampicillin: 100 µg/mL
- Tetracycline: 50 µg/mL
- Kan: 50 µg/mL
- Pour onto the petri dishes
- Mark petri dishes using the following key:
- Ampicillin: one black line
- Chloramphenicol: two black lines
- Kan: one blue line
- Glucose: one red line
Chemically Competent Cells Transformation
This protocol uses NEB Chemically Competent Cells (ie Turbo Competent E. coli High Efficiency)
- Thaw chemically competent cells on ice.
- Transfer 50 µl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
- Add 2 µl of assembled product to NEB competent cells.
- Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at 42°C for 30 seconds. Do not mix.
- Transfer tubes on ice for 2 minutes.
- Add 950 µl of room temperature SOC media to tubes.
- Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.
- Incubate plates overnight at 37°C.
Qiagen MiniPrep
For overnight cell cultures 1~5mL of E. Coli in LB medium:
- Transfer cell culture into centrifuge tubes
- Centrifuge for 5 min. at 4,000 rpm
- Discard the supernatant
- Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
- Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Do not vortex. Continue inverting the tube until the solution becomes viscous + slightly clear blue. Do not allow lysis reaction to proceed for more than 5 min.
- Add 350 µl Buffer N3 and mix immediately + thoroughly by 4-6x inverting the tube. The solution should become cloudy.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
- Apply the supernatants from the previous step to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 60 s. Discard the flow-through.
- Wash QIAprep spin column by adding 750µL Buffer PE and centrifuging for 60 s.
- Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
Protein Protocols
BIO-RAD Protein Gel
- Loading dye + DTT (if not present): in 1.5mL microcentrifuge tube, make (loading dye+DTT) by:
- Get loading dye (Laemmli Sampling Buffer in 203-14A middle drawer under ``Western Blotting Supplies")
- Get DTT (in the biology section of the desiccator in the fridge--which is on the second shelf from bottom right side).
- Mix 54mg of DTT in 950µL of loading dye.
- Determine amount of protein and buffer in each lane according to following table: Note that the protein amount CANNOT exceed 8µg per lane for 10 lane gel, and 6µg per lane for 15 lane gel. Get 1.5mL microtubes as many as needed.
- Heat the tubes for 5-10 min, and get the ladder (marked "P" at the top in the Alaska fridge, in the box "Joshi Gel ladders/dyes") out to thaw.
- While the tubes are being heated, prepare the gel:
- Take the green cap off the wells and clean the wells with water. Repeat washing about three times. Take the tape off bottom.
- Assemble the cassette with two gels or (one gel + a gel-wall). Note that the wells should face inwards.
- Put the cassette into the gel box and pour NEW Tris-gly-SDS buffer into the cassette past. The buffer level should be past the wells. Check for any leaking. If there is a leak, reassemble the cassette and try again.
- If the Tris-gly-SDS is out, mix 100mL 10X Tris-gly-SDS with 900mL of water.
- Set up the voltage machine to the desired voltage and time. For a fast run, 190V with 35 minutes could work. For slow and cleaner result, 90V with 50min could work.
- Now get the heated samples, and the ladder. Add 10µL of ladder for 15 lane gels, 15µL of ladder for 10 lane gels. Add 15µL of the sample for 15 lane gels, 30µL for of the 10 lane gels.
- Fill the box to the 2-gel mark (or 4-gel mark if running more than 3 gels) or more with OLD Tris-gly-SDS buffer. Put the cap on and hit the run button. Check for bubbles rising.
Coomassie Staining Protocol
\item Dump the Tris-gly-SDS buffer into the ``used" bottle.
\item Disassemble the cassette, set the gel down on a paper towel, and rinse the rest.
\item Fetch a box to put the gel in. Crack the gel open on all four corners and retrieve the gel; be careful not the rip, and if sticky apply water. Put it in the box.
\item Check the staining solution (50\% methanol, 40\% water, 10\% acetic acid, 0.25mg per 100mL) in the fume-hood in the back corridor.
\item If the caps have been open, or the solution is out, make a new one (500mL) by:
\item 50\% of total volume is methanol (is in the flammable cabinet in the chemistry room). 40\% of total volume is water. Use plastic graduated cylinder for these.
\item 10\% of total volume is acetic acid (is in the acid cabinet). BE CAUTIOUS WHEN WALKING CORNERS, and switches gloves after touching acid. Use GLASS graduated cylinder for this.
\item measure out and add the Coomassie to appropriate amount (0.25mg$\times$ total volume). It is located in the ``Bahamas"
\item NOTE: methanol waste must go to the special waste been under bench (bench with the pH meter). If the waste bin is full, call the number.
\item Pour the Coomassie stain into the box.
BLACaM Assays
- Set up plate reader
- Plate should be read at 405 nm
- Shake plate for 2-3 s before reading
- Calculate concentrations and amounts needed to reach a total reaction volume of 150 μL
- Possible conditions to vary
- Ca amount
- EGTA amount (removes Ca)
- Peptide type and concentration
- Other small molecule interactors
- Possible conditions to vary
- Dilute peptides down to working stock concentration (1.25 μM) with 20 mM HEPES buffer (should be room temp)
- Dilute working stock of peptides into multiple tubes to create range of concentrations required by assay
- Note that only 50 μL of the 150 μL well volume will be the peptide, so any concentration will be further diluted by 3x
- Usually no peptide is over 500 nM final concentration in well
- Dilute BlaCaM stock with 20 mM HEPES buffer
- Note that only 20 μL of 150 μL well volume is BlaCaM stock, so final concentration will be 2/15 of diluted stock
- Final concentration of BlaCaM in well should be 125 nM
- Add CaCl2 to 20 mM HEPES to create 10mM Ca concentration
- Plate mixtures in order onto mixing plate (buffer, CaCl2, EGTA, peptides, other molecules, BlaCaM)
- Must use non-binding plates (Corning 3641) for both mixing and assay plates to prevent binding of peptides and BlaCaM to plate
- 75.5 μL 20 mM HEPES + 10 mM Ca to all wells (5 mM final concentration)
- 50 μL 20 mM HEPES (no Ca) to BlaCaM only controls
- 70 μL 20 mM HEPES (no Ca) to blank (no BlaCaM, no peptide) controls
- 50 μL peptide to all but control wells
- Each concentration of peptide should be assayed at least three times
- 20 μL BlaCaM to all wells but blank controls
- Mix each well and incubate for 60 min on shaker (22-24°C)
- At 50 min prepare assay plate by adding 2-3 μL CENTA substrate
- Aim for final CENTA concentration of ~150 μM
- Add 97-98 μL of incubated mixture to assay plate and mix well
- Creates a total assay plate volume of 100 μL
- If possible, use Liquidator pipette system so all 96 wells can be transferred at once
- Immediately take read in plate reader and take reads every 2-3 min for 20 min
GLucCaM Expression
GLucCaM expression in NEB Turbo E. coli.
- Prepare 5-10mL starter culture from a fresh colony or glycerol stock in LB media supplemented with 50µg/mL CM and 0.2% glucose. Incubate at 37˚C overnight in shaker (225 rpm).
- Next day start 250mL. In 1L baffled flask, add:
- 250mL LB
- 367.5µL of CM (final conc 50µg/mL)
- 2.5mL of the cell culture grown overnight (final 1:100 dilution)
- Shake in 37˚C at 225 rpm for about 3 hours to an O.D. of ~0.6
- Induce cells with 0.4mM IPTG and place immediately into an 18˚C incubator for expression. Incubate for 14-18 hours (overnight)
- Spin down cells in large floor centrifuge for 20 mins at 3000g and 4˚C
- Save cells in -20˚C freezer until ready for purification. (OR WHO KNOWS)
DNA Protocols
Agilent Herculase II PCR Amplification
Agilent Herculase II PCR Amplification Protocol:
- In a PCR tube, mix (50 µL final volume):
- 35 µL water
- 10 µL HercII Buffer (5X)
- 0.5 µL dNTP (25mM each, 100mM total final)
- 1.25 µL forward primers (10 µM)
- 1.25 µL reverse primers (10 µM)
- 1 µL Template DNA (diluted to 10 ng/µL)
- 1 µL HercII DNA polymerase
- Give the tubes a brief spin
- Thermocycling conditions (see diagram below):
- Step 1: 95˚C, 2:00
- Step 2 X30
- Phase 1: 95˚C, 0:15
- Phase 2: (Tm - 5)˚C, 0:20
- Phase 3: 72˚C, 0:30 per 1kb
- Step 3: 72˚C, 3:00
Primer Prep
- First dilution: dilute to 100 µM (shortcut: amount_in_nMoles X10 µL)
- Second dilution: dilute to 10 µM
Gibson Assembly
For 2-3 fragment assembly
- Set up the following on ice in PCR tube (total volume 20µL):
- 0.02–0.5 pmols of fragments: 50-100ng of vectors with 2-3 fold of excess inserts.
- 10 µL of Gibson Assembly Master Mix (2X)
- Fill to 20µL with DI water
- Incubate in a thermocycler at 50˚C for 60 min.
- Wipe the lower and upper measurement pedestals with 2µL DI water
- Measure blank by pipetting 2µL of sample solvent (usually water or DNA elution buffer) on the lower pedestal
- Measure conc. of samples by 2µL on the lower pedestal
ZYMO DNA Clean & Concentrator
ZYMO DNA Clean & Concentrator - 5 Kit was used to purify DNA from PCR.
- Add 2-7 Volumes of DNA Binding Buffer to each volume of DNA sample.
- For plasmid or genomic DNA 2kb, add 2 volumes.
- For PCR or short DNA fragments, add 5 volumes.
- For ssDNA purification, add 7 volumes
- Load the mixture into a Zymo-Spin Column in a Collection Tube.
- Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.
- Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds.
- Repeat previous step, this time centrifuging for 1 minute.
- Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer or water directly to the column matrix and spin (30 seconds) to elute the DNA.
GeneWiz Sequencing Prep
The following protocol should be followed to prepare DNA for submission to GeneWiz for sequencing.
- Complete online order form.
- Label PCR tubes with the appropriate names form the order form print out.
- Concentrate template DNA according to the table below.
- Mix 10 µl of DNA template with 5 µl of primer.
- Attach PCR tubes to order form and submit GeneWiz mailbox.


text:
NEW ORLEANS — A power outage at the Super Bowl put the nation's biggest sporting event on hold for more than a half-hour Sunday, interrupting an otherwise electric, back-and-forth game that ended with Joe Flacco and the Baltimore Ravens as NFL champions thanks to a 34-31 victory over the San Francisco 49ers.
NEW ORLEANS — Joe Flacco, voted the MVP, threw three first-half touchdown passes to cap an 11-TD, zero-interception postseason. Jacoby Jones returned the second-half kickoff 108 yards, a Super Bowl record, to give Baltimore a 28-6 lead.
Moments later, lights lining the indoor arena faded, making it difficult to see. When action resumed, Colin Kaepernick and the 49ers scored 17 consecutive points, getting as close as 31-29.
But Baltimore stopped San Francisco on fourth-and-goal from the 5 with under 2 minutes left when Kaepernick's pass sailed beyond Michael Crabtree in the end zone.
The biggest deficit a team has ever overcome to win a Super Bowl is 10 points, and there were moments were it appeared San Francisco had a chance to better that mark. Instead, the 49ers lost for the first time in six trips to the Super Bowl.
Video coverage of Super Bowl XLVII
The AFC champion Ravens (14-6), a franchise that moved from Cleveland to Baltimore 17 years ago, improved to 2-0 in the big game. They also won the championship in 2001, when linebacker Ray Lewis was voted the game's MVP. Lewis was not a major factor this time, but he was a center of attention, playing in the final game of his 17-year career before retiring.
The 49ers struggled early in the first Super Bowl coaching matchup between brothers: Baltimore's John Harbaugh is 15 months older than San Francisco's Jim Harbaugh.
Baltimore led 28-6 after Jones opened the second half with the longest kickoff return in a Super Bowl, his eyes glancing up at the videoboard, presumably to watch himself sprint to the end zone. The 49ers showed they were capable of a comeback in their previous game: They trailed by 17 against the Atlanta Falcons before winning the NFC championship game.
Shortly following Jones' return, the sudden, odd power outage arrived. Escalators weren't working. Officials stopped play about 1½ minutes into the third quarter, and the bizarre delay lasted 34 minutes in real time before action resumed. Some players sat. Others stretched. Some fans chanted, "Let's go, Ravens!" Others passed time by doing the wave.
This was the 10th time New Orleans hosted the big game — tying Miami for most in a city — and first since Hurricane Katrina devastated the Big Easy in August 2005.
When play resumed, NFC champion San Francisco (13-5-1) began making things more interesting, scoring 17 points in less than 4½ minutes.
First, Kaepernick threw a 31-yard touchdown pass to Crabtree, pulling them within 15 points midway through the third quarter. Ravens defensive backs Cary Williams and Bernard Pollard missed tackles on the play. Then, with 5 minutes left in the third quarter, Frank Gore swept around right end for a 6-yard TD run, making it 28-20, before Ravens running back Ray Rice's fumble gave the ball right back to the 49ers.
San Francisco tacked on David Akers' 34-yard field goal to get within 28-23 after he missed from a longer distance but the Ravens were whistled for running into the kicker. It was his third successful kick of the game after hitting from 36 and 27 yards in the first half.
How close was it heading into the fourth quarter? Each team had exactly 17 first downs. Total yardage was nearly the same, with the 49ers slightly ahead, 317-315. Time of possession was nearly split down the middle, too.
About 2 minutes into the fourth quarter, rookie kicker Justin Tucker made a 19-yard field goal to stretch the Ravens' lead to 31-23. Not long later, Kaepernick's 15-yard run around the left side — the longest TD run by a quarterback in Super Bowl history — made it 31-29. His 2-point conversion pass intended for Randy Moss was incomplete.
A 38-yarder by Tucker made it 34-29 with 4:19 left in regulation. Baltimore purposely gave up a safety in the closing seconds to run time off the clock, setting the final score.