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HiPer Plasmid DNA Cloning Teaching Kit
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1 HiPer Plasmid DNA Cloning Teaching Kit Product Code: HTBM022 Number of experiments that can be performed: 5 Duration of Experiment: 4 days Day 1- Preparation of media and revival of E. coli Host Day2- Inoculation of host strain from single colony and ligation of insert to vector Day 3- Preparation of competent cells and transformation of the ligation mix Day 4- Observation and interpretation Storage Instructions: The kit is stable for 6 months from the date of receipt Store E. coli Host at 2-8 o C Store control plasmid DNA, vector, insert, 10X DNA ligase buffer, T4 DNA ligase and ampicillin at -20 o C Other kit contents can be stored at room temperature (15-25 o C) 1
2 Index Sr. No. Contents Page No. 1 Aim 3 2 Introduction 3 3 Principle 3 4 Kit Contents 5 5 Materials Required But Not Provided 5 6 Storage 6 7 Important Instructions 6 8 Procedure 6 9 Flowchart 8 10 Observation and Result 9 11 Interpretation 9 12 Troubleshooting Guide 9 2
3 Aim: To learn the process of cloning a foreign gene into a vector. Introduction: The cloning experiments of Herbert Boyer, Stanley Cohen, Paul Berg, and their colleagues in the early 1970s ushered in the era of recombinant DNA technology. Recombinant DNA is a DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule. The basic procedure of molecular cloning involves a series of steps. First, the DNA fragments to be cloned are generated by using restriction endonucleases. Second, the fragments produced by digestion with restriction enzymes are ligated to other DNA molecules that serve as vectors. Vectors can replicate autonomously (independent of host genome replication) in host cells and facilitate the manipulation of the newly created recombinant DNA molecule. Third, the recombinant DNA molecule is transferred to a host cell. Within this cell, the recombinant DNA molecule replicates, producing dozens of identical copies known as clones. As the host cells replicate, the recombinant DNA is passed on to all progeny cells, creating a population of identical cells, all carrying the cloned sequence. Finally, the cloned DNA segments can be recovered from the host cell, purified, and analyzed in various ways. Principle: Cloning refers to the exact copy of an organ, whole organism, single cell or a piece of DNA. Gene cloning is a process through which an exact copy of a particular gene is made. The process through which a foreign piece of DNA is transferred into another DNA led to the development of recombinant DNA technologies. The entire cloning procedure involves the following steps: 1. Isolation of pure vector and insert DNA 2. Restriction digestion of the DNAs 3. Ligation of the two linear DNA fragments 4. Transformation of the ligated product 5. Screening for the right clone 1. Isolation of pure vector and insert DNA: During cloning the foreign DNA is isolated after following the specific DNA isolation procedure. The insert DNA which contains the gene of interest is obtained after restriction enzyme digestion. Several naturally occurring plasmids have been engineered to make different types of cloning vectors. Cloning vectors are circular DNA molecules in which DNA fragments/insert are maintained and amplified. Vectors should have the following features that make them compatible for a variety of uses in recombinant DNA procedures: Size: Vectors are relatively small molecules; most are only kb in size. Ori: A vector should contain an origin of replication (ori) in order to maintain its autonomous state. Marker: To facilitate selection of cells that contain the vector, a vector should carry at least one genetic marker for which an easy assay exists (e.g., ampicillin resistance, Ampr). Cloning sites: A vector should contain several different restriction endonuclease sites that allow the insertion of a gene of interest during cloning. Many cloning vectors like puc19 contain a synthetic DNA sequence called a polylinker, which consists of 21 closely- packed RE recognition sites in a small region of DNA. 3
4 2. Restriction digestion of the DNAs:During cloning procedure an insert is added to a vector and for this reason both of them have to be cleaved by restriction endonuceases (REs). Restriction endonucleases recognize a specific, rather short, nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site or elsewhere, depending on the type of enzyme. The target site or sequence which the restriction enzyme recognizes is generally from 4 to 6 base pairs and arranged in a palindromic sequence. Generally both the vector and the foreign DNA are digested with the same RE. The restriction digested vector DNA has to be dephosphorylated to prevent self-ligation. Foreign DNA which contains the insert Foreign DNA has two restriction sites Both the vector and the foreign DNA are digested with the same restriction enzyme Cohesive ends are generated Plasmid vector Plasmid has one restriction site Cleaved DNA is ligated with the plasmid vector Fig 1: Cloning of a foreign DNA fragment into a vector involves several steps 3. Ligation of two linear DNA fragments:two linear DNA molecule ends (vector and insert) can be joined together through a process called ligation. This process involves the formation of a covalent bond between two DNA fragments (having blunt or overhanging, complementary, 'sticky' ends) by the help of an enzyme named as ligase. DNA ligase forms a phosphodiester bond between the 3 hydroxyl of one nucleotide and the 5 phosphate of another. Ligation can be directional or non-directional based upon the restriction enzyme used. When both the vector and the insert are digested with a single RE then the ligation can occur in either direction and when they are digested with two REs then ligation takes place only in one direction. 4. Transformation of the ligated product: The ligation reaction mixture is introduced into bacterial cells in a process called transformation. In this method cells are incubated in a concentrated calcium salt solution to make their membranes leaky. The permeable 4 competent cells are then mixed with DNA (ligation
5 mix) to allow entry of the DNA into the bacterial cell. Alternatively, a process called electroporation can be used that drives DNA into cells by a strong electric current. Successfully transformed bacteria will carry either recombinant or nonrecombinant plasmid DNA. Multiplication of the plasmid DNA occurs within each transformed cell. A single bacterial cell placed on a solid surface (agar plate) containing nutrients can multiply to form a visible colony made of millions of identical cells. As the host cell divides, the plasmid vectors are passed on to progeny, where they continue to replicate. 5. Screening of the right clone:the last step during cloning is screening for the right clone. Screening means to detect the right clone among a population of colonies and the screening procedure depends upon the vector used for cloning. The E. coli plasmid puc19 carries sites for different restriction enzymes (Multiple Cloning Site, MCS) within the N-terminal coding sequence for β-galactosidase of the lac operon. When puc19 is transformed into a competent host cell (which has a deletion at the amino terminal end of the LacZ gene, which codes for β-galactosidase), the truncated products from both complement each other and as a result enzymatically active β-galactosidase is produced. This is called α-complementation. The tranformants turn blue on X-gal and IPTG containing plates due to the production of β-galactosidase. X-gal is a chromogenic substrate of β-galactosidase and IPTG acts as the inducer for the expression of this enzyme. If an insert is cloned within the MCS of puc19 then β-galactosidase is not produced and as a result the transformants turn white on X-gal and IPTG containing plates. As a result one can directly screen for the insert containing vector (recombinant). Kit Contents: The kit can be used to clone GFP (Green Fluorescent Protein) gene in puc19 vector and to screen for the right clone under UV light. Table 1: Enlists the materials provided in this kit with their quantity and recommended storage Sr. No. Product Quantity Materials provided Code 5 expts Storage 1 MB104 Ampicillin 0.03 g -20 o C 2 M1245 Luria Bertani (LB) Broth 8.5 g R T 3 M1151 Luria Bertani Agar 25 g R T 4 TKC027 E. coli Host 1 No. 2-8 o C 5 TKC195 Vector ml -20 o C 6 TKC196 Insert ml -20 o C 7 TKC197 Positive Control Plasmid ml -20 o C 8 TKC198 10X Ligase Assay Buffer ml -20 o C 9 TKC199 T4 DNA Ligase ml -20 o C 10 TKC065 1M Calcium chloride (Sterile) 20 ml R T 11 PW1139 Collection Tubes, Polypropylene (2.0 ml) 18 Nos. R T 12 CG282 Polypropylene Tube (0.2 ml) 7 Nos. R T Materials Required But Not Provided: Glass wares: Conical flask, Measuring cylinder, Beaker Other requirements: Micropipettes, Tips, 50 ml Centrifuge Tubes, Water bath (42 o C), 37 o C Incubator, 37 o C Shaker, Centrifuge, UV Transilluminator, Crushed ice, Sterile double distilled water, Sterile loop and spreader Storage: 5
6 HiPer Plasmid DNA Cloning Teaching Kit is stable for 6 months from the date of receipt without showing any reduction in performance. On receipt, store E.coli Host at 2-8 o C. Vector, insert, control Plasmid, 10X ligase buffer, T4 DNA Ligase and Ampicillin should be stored at 20 o C. All other kit contents can be stored at room temperature (15-25 o C). Important Instructions: 1. Read the entire procedure carefully before starting the experiment. The entire procedure should be carried out under sterile conditions. 2. Preparation of 0.1M Calcium chloride (Sterile) (200 ml): To prepare 200 ml of 0.1M Calcium chloride, take 20 ml of 1M Calcium chloride and add 180 ml of sterile distilled water. Store at 2-8 o C. 3. Prior to the preparation of competent cells, pre-chill the tubes, 0.1M Calcium chloridesolution and centrifuge tubes. Set the centrifuge at 4 o C and water bath at 42 o C. 4. Transformation should be carried out as soon as possible after the competent cells are prepared. Storage of competent cells leads to poor or no transformants. 5. Preparation of LB (Luria Bertani) broth (55 ml): Dissolve 1.38 g of LB media in 55 ml of distilled water. Sterilize by autoclaving. 6. Preparation of LB (Luria Bertani) agar plates (20 ml): Dissolve 0.8 g of LB agar in 20 ml of sterile distilled water. Sterilize by autoclaving and pour on sterile petriplate. 7. Preparation of Ampicillin: Dissolve 30 mg of ampicillin powder in 600 μl of sterile double distilled water to give a concentration of 50 mg/ml. 8. Preparation of LB (Luria Bertani) Agar plates containing Ampicillin (100 ml): Dissolve 2.5 g of LB media and 1.5 g of agar in 100 ml of distilled water. Sterilize by autoclaving and allow the media to cool down to o C. Add 100 μl of ampicillin, to 100 ml of autoclaved LB agar media, mix well and pour on sterile petriplates. Procedure: Day 1: 1. Open the vial containing culture and resuspend the cells with 0.25 ml of LB broth. 2. Pick up a loopful of culture and streak onto LB agar plate 3. Incubate overnight at 37 0 C. Day 2: 1. Inoculate a single colony from the revived plate in 1 ml LB broth. 2. Incubate at 37 o C overnight. 3. Thaw 10X Ligase Buffer, T4 DNA Ligase, Vector and insert on ice and then set up the ligation reaction as follows: Water Vector DNA Insert DNA : 11 μl : 2 μl : 4 μl 6
7 10X Ligase buffer : 2 μl T4 DNA ligase : 1 μl Mix the contents by tapping gently and incubate at 16 C water bath, overnight. Day 3: 4. Take 50 ml of LB broth in a sterile flask. Transfer 1 ml overnight grown culture into this flask. 5. Incubate at 37 o C shaker at 300 rpm for 3-4 hours till the O.D reaches ~ 0.6. A) Preparation of Competent Cells: Note: Prepare competent cells within 3 days of reviving the strain. 1. Transfer the above culture into a prechilled 50 ml polypropylene tube (not provided). 2. Allow the culture to cool down to 4 o C by storing on ice for 10 minutes. 3. Centrifuge at 4100 rpm for 10 minutes at 4 o C. 4. Decant the medium completely. No traces of medium should be left. 5. Resuspend the cell pellet in 30 ml prechilled sterile 0.1 M Calcium chloride solution. 6. Incubate on ice for 30 minutes. 7. Centrifuge at 4100 rpm for 10 minutes at 4 o C. 8. Decant the calcium chloride solution completely. No traces of solution should be left. 9. Resuspend the pellet in 2 ml prechilled sterile 0.1M Calcium chloride solution. 10. This cell suspension contains competent cells and can be used for transformation. B) Transformation of cells: 1. Take 200 l of the above cell suspension in three 2.0 ml collection tubes and label them as control, plasmid and ligation mix. Add 2 l of positive control plasmid DNA to the tube labeled as plasmid and mix well. Add 10 l of the ligation mix to the tube labeled as ligation mix and mix well. 2. Incubate all the 3 tubes on ice for 30 minutes. 3. Transfer them to a preheated water bath set at a temperature of 42 o C for 2 minutes (heat shock). 4. Rapidly transfer the tubes on ice. Allow the cells to chill for 5 minutes. 5. Add 800 l of LB Broth to all the tubes. Incubate the tubes for 1 hour at 37 o C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. 6. Plate 200 l of cell culture from each tube on LB agar plate containing ampicillin using a sterile spreader. 7. Store at room temperature till the plates are dry. 8. Incubate the plates overnight at 37 o C. 7
8 Flowchart: 0bservation and Result: After incubation observe the plates under UV light (on a UV transilluminator) and look for the glowing green colonies on the plasmid plate. For the ligation mix plate look for both glowing and non-glowing colonies. Interpretation: On transformation of cells with positive control plasmid, antibiotic resistance is conferred on the host as this plasmid carries gene for ampicillin resistance. As a result, those cells that grow in presence of ampicillin are transformed cells. In contrast, no growth is observed on plating competent cells untransformed with vector DNA as the host E.coli cell is sensitive to ampicillin. The transformed colonies of the positive control plate glow when exposed to UV light as it contains GFP gene. The unique 3 dimensional structure of the GFP releases energy in the form of visible green light on exposure to UV light. For the ligation mix plate, the colonies which carry the recombinant plasmid (vector+insert) glow under UV light and those which 8
9 carry only the vector (self-ligation) do not fluoresce under UV light. Troubleshooting Guide: Sr. No. Problem Possible Cause Solution 1 Transformants from the ligation mix do not fluoresce under UV light Ligation is not done properly Set up the ligation assay as mentioned in the protocol. Always thaw the ligase buffer and the enzyme on ice. Try to maintain the temperature during ligation Cold chain not maintained properly during the procedure Always keep the cells on ice & make sure the centrifuge machine is cold throughout the experiment 2 Few or no transformants Heat shock treatment not done accurately Ampicillin concentration used is not correct Follow the procedure, with exact time duration for heat shock treatment While making the plates add accurate amount of ampicillin Plates were not incubated at proper temperature for sufficient time Incubate the plates at 37 o C for minimum 18 hours 3 Observed bacterial growth on control plate Ampicillin is inactivated Add ampicillin to the media after it cools down to o C 4 Contamination observed on plates Sterility not maintained during the experiment Make sure that the entire procedure is performed aseptically Technical Assistance: At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of technical assistance, mail at PIHTBM022_O/0117 HTBM
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To quickly assess the level of stakeholder impact and commitment to a change programme and develop an outline plan for engagement.
Having a critical mass of committed stakeholders is essential for the success of a change programme. Developing a plan for engagement must go beyond the conventional communication activities and consider ways of actively engaging stakeholders to achieve real commitment.
The 'Stakeholder Engagement' Solution helps identify the key stakeholders that will affect success and develop appropriate strategies that translate to a managed programme of ongoing engagement.
This solution can be utilised at any stage of a change programme and improvements can be realised quickly. Ideally, it should be conducted once the impact of a planned change is known in order to inform the stakeholder analysis.
Working with the team responsible for implementing the change, we first identify the stakeholders and assess their importance to the programme. We then prioritise the stakeholders and agree a set of generic engagement strategies that builds on existing work and plans.
We then build collaboratively an Engagement Plan that outlines the planned engagements, the expected impacts and responsibilities for delivering it. We also help establish an agreed set of engagement principles and roles to ensure the organisation has the capability to deliver the Plan.
The Outputs that are delivered by the 'Stakeholder Engagement' Solution are:
We have an example of a Stakeholder Map in the right-hand panel - move your cursor over the Map to see the level of detail produced.
If you want to discuss this service in more detail click here and we'll be in touch.
The two main outcomes of the 'Stakeholder Engagement' Solution are the Stakeholder Map and the Engagement Plan; simplified examples of each are shown below.
The Stakeholder Map helps to formulate generic engagement strategies e.g. stakeholders in the upper right quadrant warrant high levels of engagement as they have a high interest in the programme and are very influential.
Hover with your mouse to zoom in:


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Fort Mill Times
John Queen and Jennifer Sweeney of Indian Land are the parents of a son, Andrew Parker Queen, born April 17, 2012.
Maternal grandparents are Eric Sweeney and Donna Burgee, both of Indian Land.
Paternal grandparents are Robby and Rosewitha Queen of Lockhart.
Thomas and Amanda Walters of Fort Mill are the parents of a son, Jonathan Mikael, born April 13, 2012.
Maternal grandparent is Terri Sterling of Jacksonville, N.C.
Paternal grandparent is Tammy McSon of Fort Mill.
Victor Worley and Krista Millwood of Fort Mill are the parents of a daughter, Isabella Grace Worley, born April 17, 2012.
Maternal grandparent is Sara B. Millwood of Fort Mill.
Paternal grandparents are D.D. and Roy Worley of Rock Hill.


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Climate change, simply put change in global weather patterns, is unarguably the biggest environmental issue of our time. Climate change is global in its causes but its consequences are far more reaching in developing countries particularly Nigeria whose biodiversity and ecosystems are already faced with other threats like habitat degradation, ecology-economics mismatch and escalating population growth.
NCF acknowledges that climate change is the most important human-induced environmental challenge that could exacerbate biodiversity loss caused by habitat destruction, invasive alien species and the like. We have therefore put in place 5-point mitigation and adaptation strategies to combat climate change in Nigeria. These are enlightenment campaign, mitigation strategy, adaptation initiatives, policy and advocacy, and carbon finance.
1. Enlightenment Campaign
Results of climate change awareness survey recently conducted by British World Service Trust revealed that an average Nigerian is not aware of climate change despite its impact on our day-to-day activities. Little or no attention is paid to alarming change in weather pattern such as excessive flooding experienced in different parts of Nigeria in 2008, increased aridity and intense desert encroachment witnessed in northern Nigeria in the past one decade or the dwindling flow of Niger Basin and other major waterways corroborating poor agricultural yield, communal clashes over natural resources management and siltation of the river basin.
NCF therefore has in its on-going climate change enlightenment campaign strategies to sensitize Nigerians, particularly youth across the country and women in rural environment who are the most vulnerable groups to climate change and its attendant impact. The enlightenment campaigns will contribute to climate change adaptation strategies and address what Nigerians can do as a citizen to lessen the impact of climate change.
The pilot phase of the enlightenment campaign started in 2008 with the annual celebration of World Environment Day (WED) 2008 tagged CO2: Kick the Habit. In tandem with the celebration, the enlighten campaign was also mainstreamed into the toolkit of NCF facilitated School Conservation Clubs. The School Conservation Clubs spread across the country is an arm of Education Unit of NCF instituted to reach out to and empower secondary school on environmental sustainability issues.
2. Mitigation Strategy
Tropical forests accounts for 15% of the earth land cover and contains not less than 25% carbon stock on earth surface. Despite the significant role of carbon storage among other vital benefit to man's existence, they are daily unsustainably deforested with little or no regeneration plan. Deforestation thus leads to emission of carbon dioxide, major constituent of Greenhouse Gases into the atmosphere making change in land cover second largest contributor to global warming.
Although Nigeria may not have the finance resources or sophisticated technology to mitigate climate change, the country is endowed with mosaic of natural ecosystems that have the capacity for carbon sequestration. NCF therefore plans to combat climate change in Nigeria through:
i) Implementation of Reducing Emission from Deforestation and Degradation (REDD) for Protected Area management;
ii) Development of REDD Action Plan (REDDAP) for Nigeria in collaboration with relevant Government agencies, private sector and civil societies;
iii) Engaging in climate change policy discussions especially on REDD;
iv) Scientific research on carbon stock assessment/forecast.
3. Adaptation Initiatives
Climate change poses development setback to Nigeria. With increasing global warming impact, wet areas are experiencing intermittent flooding and dry areas getting drier. Consequently, ecosystems and biodiversity which underpin biological productivity and socio-economic development, through the provision of many goods and services to people and the manufacturing sector will eventually be degraded if urgent steps are not taken.
Ecosystem services produced by ecosystems and biodiversity such as food, water, timber, fuel and fibre, particularly food are one of the key drivers of inflation rate. Scarcity of or shortage in supply to the food market due to poor agricultural yield triggers high cost of essential commodities which has significant impact on the nation's economy. It will suffice to say climate change will exacerbate the dwindling inflation rate of essential commodities required for daily living.
NCF climate change adaptation initiative therefore seeks to assist communities especially those around our intervention sites to increase their resilience to climate change induced challenges such as food security, agriculture, watershed management, and natural systems (i.e. ecosystems and biodiversity) conservation
4. Policy Advocacy
Nigeria is yet to be at the forefront of climate change policy forum at regional and international levels despite the alarming effect of changing climate on lives and livelihoods across the 6 geo-political zones of Nigeria. We are constrained by resolution of international agreement that we are party to despite little or no technical input to adopted positions.
Hence, NCF plans to gather relevant technical information required for holistic climate change policies in Nigeria and also support Nigeria's position at regional and international climate change forums.
5. Carbon Finance
Several funding schemes have been adopted globally to support climate change mitigation and adaptation such as Clean Development Mechanism (CDM) under Kyoto Protocol. Nigeria however is barely able to benefit from these global resources due to several factors such as stringent CDM funding guideline and inadequate private sector engagement in climate change programmes.
Going forwards, NCF plans to raise funds to support climate change projects through 1% support for climate change campaign. The campaign seeks Government injunction for private sector to contribute 1% net profit to fund climate change agenda in Nigeria. Individuals will also have opportunity to offset their carbon footprint and join the global league of carbon neutral scenario.
NB: Press Cutting Service
This article is culled from daily press coverage from around the world. It is posted on the Urban Gateway by way of keeping all users informed about matters of interest. The opinion expressed in this article is that of the author and in no way reflects the opinion of UN-HABITAT.


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Music Player MP3 Player, Audio Player 220.127.116.11 APK Unlocked
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Searching for the best choice for toenail fungus treatment? Perhaps you may have been serious to choose the best treatment for stopping nail fungus. You might have been frustrated due to the nail fungus getting been not wiped out in many treatment solutions. Your frustration is pretty significant as there are several choices which can be no longer working properly setting your final strategy to your disorder. I will know what your brain anticipates. Perhaps you seek a rapid and permanent remedy for nail fungus.
Most of the remedies introduced through latest prescribed centered medicines with contra- yeast outcome are not doing work well to 100% certainty. In many cases, the yeast illness has opened up the dark door again for repeat. What is the fundamental reason behind the issue of recurrence inspired of the best remedy you might have opted? Have you thought of the problem? It might possibly be the way it is of absence of Follow Up cures to get rid of the onycosolve price entirely with the very least possibility of repeat.
Getting satisfactory care to safeguard the healthier toenail progress is essential. Once the ingrown toenail fungus has been destroyed completely, you should concern about avoiding re-disease through the days and nights to come. Though it may be not transmittable to some foreign physique, the contagiousness will work inside the identical entire body if appropriate Follow Up care will not be delivered to steer clear of the exact same. The cured toenail might have repeat as a result of a lot of reasons like humid floor or unclean maintenance of the finger nail. It is merely a good perception to remove toenail fungus permanently be preserving your healthy nail protected against all probably possibilities for re-infection to your agony. The main thing you ought to concern is the shaping your finger fingernails regularly.
It is actually a forewarning for you that you ought to care for day-to-day foot cleaning with many anti fungus detergent or option. Using anti-bacterial applies inside the footwear and socks could be letting you retaining off illness from recurrence. Keeping healthier fingernails when you eat alkaline meals is likewise suggested even with dealing with your infected nail. Even though you could not have a comprehensive power over toenail fungus, there is crash chance to shield your wholesome nail from becoming re-contaminated from now on. You will find home cures with home goods like vinegar, green tea tree gas, lavender oil, Oregano oils, and essential olive oil. Between these, Vinegar is really a natural alkaline item to take care of toenail fungus as do-it-yourself solution. The remedy is effectively working to avoid and do away with nail fungus.


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Is civilization merely
cooperative predation,
supporting illusions
of physical escape
from consequences
of eating and
being eaten?
Our actions speak clearly
of sly dissimulation
as deliberate confusion
offers alluring bait
to multiple chances
for cheating and
being beaten.
Then why do I love so dearly
this language of elucidation
unless by poetic infusion
I challenge to reshape
financial romances
into weeding and
being seeded
with genetic discpline yearly
self-limit each population
to end our wild profusion
and, hoping it's not too late,
save children of gentle sciences
for nurtured loving and
being needed.
next poem
previous poem
Storyline Index page 1, 2, 3
Alphabetical Index page 1
Angelfire Home Pages
Absolute Background
Textures Archive
John Talbot Ross


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Welcome back to lesson 3 – Defining Content Structure. Hopefully you have had a chance to run through the work book and have begun to think about the amazing content you will be creating for your readers/viewers.Continue reading
Have you jumped ahead? If so you can hop on over to the first session here…. On to session number 2, Finding a Platform.Continue reading
I'm really excited about this series to create an income stream from your daily thoughts, or "Blogging for Profit". I know I'm always excited about my new course materials!Continue reading
Upcoming topics include: What's a Blog and How Does It Work? Finding a Platform Defining Your Content Structure Legal Stuff and What You Need How to Install WordPress How to Write a Blog Post on WordPress Themes and Why I Recommend Thrive Magic of Media (for each post) Planning Your Content (checklist) How to Monetise […]Continue reading
Hiya, Fellow Yarn Lovers! If you have a WordPress blog, there's a nifty little trick that I think you might like. I'll show you how to do it, right here in the Academy! For those of you who've spent a fair bit of time on Pinterest (you know who you are!), you know that there's […]Continue reading


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Sola is a Tipperary based Energy Solutions provider and installer. The company was established in 2008 and is run by its managing director, Paul O'Brien.
The company was initially established as a solar panel supplier and installer. However as new technologies and products with increased outputs and efficiencies became available, the company expanded its portfolio to include these various energy systems.
The company now supplies and installs a variety of high quality systems which include:
Our portfolio of works completed includes:
Our working principal is to ensure we only use the best resources and materials. Over the years we have worked to establish solid working partnerships with company's such as Mitsubishi Electric, Joule, LVP, Conergy and many others to ensure our customers receives the highest quality product & service.
We have also worked closely and progressively with SEAI to keep up to date with progress in the Irish economy. We try, where possible, to use Irish & local manufactured products and suppliers.
We are fully trained and registered installers under the greener homes scheme, and have over 800 solar installations across Ireland.
The Sola Team are all fully trained in the required areas of solar and heat pump installations & Stove Fittings. This ensures that when our team of professionals arrive on site the job is completed in a timely manner.


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Cheap Tour Sydney
- Return International Airfare and airport taxes
- 3 nights' accommodation in Melbourne
- 3 nights' accommodation in Sydney
- Daily Breakfast
- Internal Airfare (Melbourne – Sydney)
- Return Airport transfers in Melbourne and Sydney on seat in coach basis
- Choice of any 3 attractions: Sea life Sydney Aquarium, Wildlife Sydney Zoo, Sydney Tower Eye, Manly Sea life Sanctuary or Madame Tussauds)
- Australia Visa Charges
- Travel Insurance up to age of 60 as per tour duration
- Costs of meals on internal domestic flights are not included in the tour price.
- The package does not include any expenses of personal nature such as laundry, wines, mineral water,
- Food and drinks and other things not mentioned in our itinerary.
- The package cost does not include porter services at hotels, tips, or insurance charges.
- Any Meals apart from what is mentioned in your itinerary.
- Cost of excursions, sightseeing tours, entrance fees and local guides availed of by the passengers, other than what is mentioned in the package inclusions
Day 1 – Arrival in Melbourne
Shabby Tour In Sydney Land at Melbourne Tullamarine Airport, trade (arrange in-tutor preface) to the cabin and enrollment for a medium-term remain.
Melbourne, the second greatest city in Australia, experiences a smooth oceanic environment. The uncommon inclination of this exuberant city is made by the blend of street workmanship, rooftop bars, diners and cosmopolitan lifestyle. Frequently called the social capital of the country, Melbourne has stacks of craftsmanship presentations, exhibitions, theaters, demonstrate passages and open libraries.
Day 2 – Half-day City Tour (Optional) | Visit to Phillip Island (Optional Tour)
Post breakfast, set out on a half-day city voyage through Melbourne.
Arranged in the south-eastern corner of territory Australia, it is a lively city stacked with diners, cool bistros, eminent craftsmanship showcases and hip rustic regions. Melbourne has been met with the title of the Most Liveable City in the World. It has distinctive get-away spots like Federation Square, Queen Victoria Market and Royal Botanic Gardens.
After the city visit closes at the Federation Square, leave for Phillip Island.
Phillip Island (Optional Tour), found around 140 km from Melbourne, is a guest objective with more than 3 million people going to it consistently. Well off in untamed life, this island gives adequate spots to whale and fowl seeing. It incorporates some stunning shorelines that are outstanding for practices like surfing and swimming. While you are here, see koalas, kangaroos and penguins in their typical living space.
A while later, set out back to Melbourne and accomplish the motel for some rest.
Day 3 – Great Ocean Road Adventure Tour (Optional Tour) on self drive or sharing guide
Have a tasty breakfast, and after that go out for an excursion on the Great Ocean Road.
Magnificent Ocean Road is around 240 km long, stretching out along the south-eastern shoreline of the country. Worked by the officers as a recognition to the holy people of World War I, this long road partners places like Lorne, Apollo Bay and Port Campbell. On the Great Ocean Road Adventure Tour, acknowledge wonderful points of view of the Pacific Ocean, and see attractions like Twelve Apostles and Loch Ard Gorge.
After the long and splendid excursion, wander out back to the cabin and rest.
Day 4 – Depart Melbourne and travel to Sydney
Land at Sydney Airport, trade (sharing coach preface) to the hotel and enlistment for a medium-term remain.
Sydney is the state capital of New South Wales that features the world's greatest trademark harbors and acclaimed explorer objectives like Sydney Opera House and Harbor Bridge.
Upon section in Sydney, trade to the hotel on sharing coach preface. Enlistment at the hotel, and welcome a not too bad night's rest.
Day 5 – Sightseeing in Sydney | Visit to Sea Life Aquarium, Wildlife Sydney Zoo, Sydney Tower or Madame Tussauds
Have a brilliant breakfast and subsequently go out for a visiting visit.
At the start of the day, you can acknowledge choice of any 3 attractions: Sea life Sydney Aquarium, Wildlife Sydney Zoo, Sydney Tower Eye, Manly Sea life Sanctuary or Madame Tussauds)
Accomplish King Street Wharf close Darling Harbor without any other individual and exchange Combo 3 Pass voucher for admissions to Sea Life Aquarium, Wild Life Sydney, Madame Tussauds, and Sydney Tower Eye.


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Las Vegas, NV – R&B vet Tank lately apologized for letting down the present era of singers however JR Castro isn't lacking a beat.
The dependable R&B savant is giving Cuffin Season '18 an ember for the fireplace with the discharge of his "Bon Voyage" video, a collaboration with musical duo, Breakfast N Vegas.
Watch the HipHopDX-premiered "Bon Voyage" video up above and observe @jrcastrolv on Instagram as he preps extra music.


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04 Aug Congratulations Dr. Knipping!
Posted at 19:11h in Dr. Knipping's Research Blog, Exercise & Fitness, Healthy Lifestyle
HUGE CONGRATULATIONS to our Medical Director, Dr. Randy Knipping. Yesterday he won the Canadian Masters 10000 m championship in Surrey BC yesterday taking home the gold medal for Toronto West Athletics in a time of 43:21.
Dr. Knipping knows all about setting goals and working hard to achieve them. Be sure to share yours with him when you're in the clinic next!


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Views: 4445 Date: 2018-04-03 Favorited: 14


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Contaminated GR gas station gets grant for redevelopment
GRAND RAPIDS — A contaminated former gas station property in Grand Rapids will be redeveloped after being receiving brownfield funding from the Michigan Department of Environmental Quality.
Reacting to consumer demands, Meijer ramps up tech initiatives
DETROIT — Meijer Inc. largely functions as a brick-and-mortar operation, but the supercenter retailer increasingly is taking cues from tech companies as it implements new ways of serving customers.
Nonprofits turn to food businesses for revenue streams
Cookies made by volunteers and residents of Degage Ministries are providing people with tasty treats and helping the organization access much-needed funding.
MiBiz Growth Report: Sept. 17, 2018
MiBiz Growth Report: September 4, 2018
Mercy Health deal for medical group expands presence in growing Ottawa County
GRAND HAVEN — A deal for North Ottawa Community Health System's medical group raises Mercy Health's presence in the growing Ottawa County market where it's been investing for a few years.
West Michigan grocery retailers shift to tech-driven marketing initiatives
Spurred on by changing consumer shopping habits, West Michigan grocery retailers want to make the task of shopping less of a "chore" by leveraging new technology-driven initiatives.
Spectrum Health pilots retail clinic at new Hudsonville Meijer store
HUDSONVILLE — The newest Meijer Inc. store in West Michigan includes a first for the region: a retail-based medical clinic operated by Spectrum Health.
Brewery execs question craft beer's emerging two-tiered pricing strategy
Stroll through the beer aisle at some West Michigan grocery supercenters and you might notice a bit of gold tucked between the red, white and blue cases of Budweiser and MillerCoors brands.
Q&A: Stacie Behler, Meijer Inc.
Stacie Behler wants to see more women in top leadership roles at West Michigan companies and nonprofits. The Meijer executive in charge of public affairs and corporate communications — and the retailer's former senior legal counsel — was recently honored by the Michigan Women's Foundation with its Woman of Courage and Achievement award. The statewide organization focuses on accelerating women's entrepreneurship, developing the next generation of women leaders and furthering policy issues relevant to women in the state, including a campaign to end the backlog of untested rape kits in Detroit. In accepting the award, Behler put particular emphasis on the mentoring and development of young women and nurturing more female business leaders. She discussed the topics before a crowd of more than 800 people in mid April when she received the award, as well as in an interview with MiBiz following the luncheon ceremony.


text: OSU football Day Got Nearly 500k Filling In For Meyer By Associated Press • Sep 20, 2018 si.com Ohio State University assistant football coach Ryan Day earned 487 thousand dollars while filling in during coach Urban Meyer's three-game suspension. Meyer Issues Statement To Clarify 'Incorrect Media Reports' By Associated Press • Sep 1, 2018 usatoday.com Ohio State head football coach Urban Meyer is pushing back on the reasons for his three-game suspension with claims that don't fully square with details laid out in the investigation that led to his punishment. OSU Trustee Resigns Over Meyer's Short Suspension By Associated Press • Aug 30, 2018 An Ohio State University trustee who thought football coach Urban Meyer deserved more than a three-game suspension has resigned from the board. OSU Allowed Meyer To Meet With Football Team After He Was Suspended By Associated Press • Aug 29, 2018 usatoday.com Ohio State football coach Urban Meyer's suspension and previous paid leave have restricted him from talking football with his staff and athletes during August with the exeception of a team meeting the day after the suspension was announced. Attorneys Say If Meyer Deleted Text Messages, It's Illegal By Associated Press • Aug 27, 2018 solveyourtech.com Open records experts say any attempt by Ohio State football coach Urban Meyer to eliminate older text messages from his phone to hide information would be without question illegal. Meyer Issues Apology To Courtney Smith By Jim Letizia • Aug 25, 2018 Associated Press Following a firestorm of criticism, suspended Ohio State University head football coach Urban Meyer has issued an apology to the ex-wife of the former assistant coach who she says abused her. Ohio State Suspends Urban Meyer And Gene Smith For Handling Of Zach Smith Allegations By Jim Letizia & Associated Press • Aug 23, 2018 Associated Press Ohio State University has suspended head football coach Urban Meyer for the first three games of the season. OSU Trustees To Meet About Meyer On Wednesday By Associated Press • Aug 21, 2018 youtube.com Ohio State University trustees have scheduled a private meeting Wednesday to talk about the future of football coach Urban Meyer. Courtney Smith Meets With OSU Investigators As Zach Smith Admits Hiding OVI Bust By Jim Letizia • Aug 14, 2018 youtube.com The former wife of fired Ohio State assistant football coach Zach Smith spoke with university investigators Monday. Courtney Smith Says OSU Never Contacted Her About Domestic Abuse Allegations By Jim Letizia • Aug 8, 2018 si.com The attorney for the former wife of fired Ohio State assistant football coach Zach Smith says the school never contacted her to discuss the allegations of domestic abuse against him. Some OSU Fans Rally To Save Meyer By Jim Letizia • Aug 7, 2018 More than 150 people stood outside Ohio Stadium last night in support of OSU football coach Urban Meyer. OSU Hires Outside Firm To Investigate Meyer By Associated Press • Aug 6, 2018 youtube.com Ohio State University says an outside firm has been hired to conduct the investigation into football coach Urban Meyer's handling of a domestic abuse complaint against a former assistant. Meyer Says He Followed Protocol For 2015 Abuse Allegations By Associated Press • Aug 3, 2018 usatoday.com Ohio State University head football coach Urban Meyer insisted Friday he followed proper protocols in reporting domestic violence allegations against an assistant coach in 2015, passionately defending himself in his first extensive comments since he was put on paid leave amid a university investigation. OSU Appoints Commitee To Investigate Meyer By Associated Press • Aug 3, 2018 si.com Ohio State University trustees have appointed a committee to investigate head football coach Urban Meyer's handling of fired assistant coach Zach Smith's domestic abuse allegations. Urban Meyer On Leave During Investigation Of Assistant By Associated Press & Alison Holm • Aug 1, 2018 cleveland.com Ohio State University has placed head football coach Urban Meyer on paid administrative leave while it investigates claims that his wife knew about allegations of domestic violence against assistant coach Zach Smith years before he was fired last week. OSU Fires Assistant Football Coach Amid Domestic Violence Allegations By Jim Letizia • Jul 24, 2018 youtube.com Ohio State University has fired assistant football coach Zach Smith after Smith's former wife filed a civil protection order against him last Friday and an allegation of previous domestic violence surfaced. Former Buckeye Kudla Passes Away By Associated Press • Jul 18, 2018 nbcsports.com Former Ohio State defensive end Mike Kudla has died at the age of 34. Former Buckeye Barton Indicted For Injuring Sheriff's Deputy By Associated Press • Jun 24, 2018 alumnigroups.osu.edu Former Ohio State University football player Kirk Barton has been indicted on charges of slamming the arm of a Delaware County Sheriff's deputy with a van door. Memorial Tournament Co-Founder Passes Away By Jim Letizia • Jun 13, 2018 facebook Funeral services are pending for Memorial Tournament co-founder Pandel Savic, who has died at the age of 92. Former Buckeye Barton Charged With Assaulting A Cop By Associated Press & Jim Letizia • Jun 5, 2018 alumnigroups.osu.edu A former Ohio State University offensive linesman has been charged with assaulting a police officer outside of a Dublin-area bar last Friday. Public Service Scheduled For Bruce By Jim Letizia • Apr 21, 2018 Associated Press A public service will be held next week following the death of former Ohio State football coach Earle Bruce. Family Announces The Death Of Former OSU Football Coach Earle Bruce By Associated Press & Jim Letizia • Apr 20, 2018 theozone.net Former Ohio State football coach Earle Bruce has died at his central Ohio home at the age of 87. OSU's Meyer Getting Contract Extension By Jim Letizia • Apr 6, 2018 cleveland.com An Ohio State University Board of Trustees' committee has approved a two-year contract extension for football coach Urban Meyer. OSU's Jim Stillwagon Passes Away By Jim Letizia • Feb 5, 2018 pinterest.com Funeral arrangements are pending for Ohio State football legend Jim Stillwagon, who passed away over the weekend at the age of 68. OSU Got More Money From Beer Sales In Ohio Stadium This Past Season By Associated Press • Dec 12, 2017 thelantern.com Records show Ohio State University collected 1.35 million dollars in net revenues from beer sales at Ohio Stadium during this past football season. AP Obtains Memo Of Understanding Signed Before Failed Greg Schiano Deal With U.T. By Associated Press • Dec 12, 2017 youtube Former University of Tennessee athletic director John Currie and Ohio State defensive coordinator Greg Schiano had signed a memorandum of understanding to make Schiano the school's head football coach before the deal fell apart amid a public backlash. Former Buckeye Cited For OVI, Posession Of Drug Paraphernalia By Associated Press & Jim Letizia • Oct 22, 2017 Westerville Police Franklin County Sheriff's deputies have cited former NFL and Ohio State quarterback Troy Smith for possession of drug paraphernalia and operating a vehicle under the influence. Ex-Buckeye Files Paperwork To Run For Congress By Associated Press • Sep 3, 2017 Getty Images A former Ohio State football player has filed the paperwork to run for Congress next year. Judge Clears Former OSU Football Player Of A Weapons Charge By Associated Press • Aug 29, 2017 cleveland.com A Hamilton County judge has acquitted former OSU and current Buffalo Bills defensive tackle Adolphus Washington of a weapons charge. Daughter Says Former Buckeye Football Coach Has Alzheimer's Disease By Jim Letizia • Aug 28, 2017 sportingnews.com 86-year-old former Ohio State University football coach Earle Bruce is the fifth member of his family to be diagnosed with Alzheimer's disease. Pages1 2 next › last »


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We will be unable to receive grain Thursday, May 16th, Friday, May 17th, and Monday, May 20th due to maintenance. We will be back receiving grain Tuesday, May 21st. We apologize for the inconvenience and appreciate your patience! Check out the Construction tab to see updates on our new Fertilizer Plant in New Rockford! We are accepting all commodities!!!!
We are offering free Delayed Pricing on Corn, Soybeans, and Spring Wheat with a pricing date of July 31st, 2019
Dumping Hours: M-F 8:00 to 5:00 *Subject to confirmation and change*
All prices based Niles; Station differentials to apply Please make sure your trucks are cleaned from fertilizer and treated seed before delivering grain to the elevator. Any contaminents in the load and it will be rejected.
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Fungo bats are usually 36 or 37 inches long. Due their thinner, lighter design, they are a very versatile tool for any coach. It is so much easier to hit hundreds of grounders and fly balls with a fungo than with a regular bat. As well, with the prices of today's top end bats, a $35 fungo makes more sense. Don't dent the $350 war clubs in your bat bag.
While many people view the fungo bat more as an antique piece of baseball's history, there's no doubt that it still has a special place in the modern game. Actually, the bat is starting to enjoy some revival all-round the game. Regardless of minimal production of fungo bats, you can rest assured that they're here to stay. The bats are too beneficial to both coaches and players on a daily basis.
Though entertaining enough, this play appears in its entirety as a small part of Sorrentino's magnum opus MULLIGAN STEW. You can read it as easily there as in this form. Plus you'll have the rest of Mulligan Stew as well. So I recommend you only buy this volume if you're the sort of collector who owns books because of what they are, not what they say.
Within league standards there is ample latitude for individual variation, many batters settling on their own bat profile, or one used by a successful batter. Formerly, bats were hand-turned from a template with precise calibration points; today they are machine-turned to a fixed metal template. Historically significant templates may be kept in a bat manufacturers' vault; for example, Babe Ruth's template, which became popular among major-league players, is R43 in the Louisville Slugger archives.[citation needed]
Fungo golf is a game involving two or more players in which they use fungo bats and baseballs and their object is to hit certain objects at a field. Fungo golf is played very similar to golf, where the player with the fewest strokes after a certain number of holes (typically nine or eighteen) wins the game. While there is no concrete history of how fungo golf was created, it has spread throughout America as a popular game especially with baseball players and coaches.


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The bride and her close friends can browse through wedding apparel designers and collections using glossy pictures too beautiful and substantial as in any published catalog. It's not just the marriage dresses that may be analyzed, possibly. There are prom dresses and mother of the bride attire which could also be thoughtfully considered on line and thoughts a lot more readily shared with friends and relatives far out of home.
What is different beneath the weddingdress is also of overriding importance, and specialty under garments can be selected and arranged online. From stockings to lingerie, to garters, and shoes to suit can be inspected along with selected online, delivered into the bride's house to be reviewed in her own convenience. The bride may additionally examine and arrange a wide assortment of headpieces, veils, and tiaras in order to discover which would go best with her hairstyle and apparel.
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The internet has revolutionized so much of our world, by your sharing of news and information, to the distribution of products and companies, however not before has it become easier to utilize the web to save time and energy in creating an absolutely perfect wedding day.