Team:Reading/Protocols

From 2014.igem.org

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<H1 style="font-color='red', text-align=center"><font-color=red>DO NOT EDIT THIS PAGE. GO TO THE GOOGLE DOC</H1>
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<!--Protocols  -->
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<tr><td><h3><font color="#558e2b"> A note on protocols </font></h3></td>
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<td> <h3 id="intro"><font color="#558e2b"> Contents </font></h3></td>
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<!-- Introduction to the Protocols Page -->
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<p><font color="#292929">
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Below are a number of examples of protocols - modified and non-modified - used throughout our project. The protocols we list below may not have been followed exactly in each instance of usage, often due to time constraints - beginning an experiment on one day and concluding it on another.
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<!-- Contents - add stuff here to add to the contents page -->
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<ol>
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<li><a href="#intro">A Note on Protocols</a></li>
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<li><a href="#protocols">The Goods</a></li>
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<ul>
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    <li><a href="#exampleone">Example one</a></li>
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    <li><a href="#exampletwo">Example two</a></li>
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<li><a href="#acknowledge">Acknowledgements</a></li>
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<li><a href="#references">References</a></li>
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<!-- The Protocols -->
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<td colspan="3"><h3 id="protocols"> <font color="#558e2b">The Goods</font> </h3>
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<p bgColor=B2E592><font color="#292929">
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Here are all of the protocols.
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<!-- PCR -->
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<p id="pcr"><font color="#558e2b"><i>PCR</i></font></p>
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PCR was used as a means of amplifying each one of our biobrick constructs. Each construct, along with its corresponding flanking sequence of 50-100 bases, was amplified out of each transformed pSB1C3 plasmid.
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Prior to PCR, it was ensured that all DNA obtained from miniprep was of a concentration of at least 1ng/ul per 100bp; this was performed using a desktop ThermoScientific NanoDrop machine. All PCR tubes were kept on ice prior to usage.
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2μl of each of the VFR (forward) and VR (reverse) primers were added to sterile PCR tubes, along with 2μl of each respective transformed plasmid and 14μl phusion mastermix [add reference here?].
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The PCR program used was as follows:
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<b>Start:</b>
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<li> 95C/30s </li>
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<b>35 cycles:</b>
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<li> 95C/10s </li>
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<li> 56C/15s </li>
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<li> 72C/70s </li>
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<b>Final:</b>
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<li> 72C/5min </li>
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<li> 68C/10min </li>
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All PCR products were cleaned up using a ThermoScientific GeneJET PCR Purification Kit, using the provided protocol<sup>1</sup>.
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<p id="ferricyanideassay"><font color="#558e2b"><i>Potassium ferricyanide assay</i></font></p>
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Another potential protocol
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<!-- Example Protocol 1 -->
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<p id="exampleone"><font color="#558e2b"><i>Isolation of plasmid DNA from bacteria (miniprep)</i></font></p>
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  <li>Pellet bacterial cells by centrifuging 1.5 ml of culture in a 1.5 ml microcentrifuge tube at 4000 rpm for 2 minutes
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  <li>Discard supernatant by pipetting off ensuring not to disturb the pellet
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  <li>Resuspend in 250 µl of resuspension solution by vortexing or pipetting up and down. Do not incubate for more than 5 minutes
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  <li>Add 350 µl of neutralisation solution and mix by inverting the tube 4-6 times
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  <li>Centrifuge at 13,000 rpm for 5 minutes
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  <li>Transfer supernatant to a GeneJET spin column by pipetting. Do not disturb the white precipitate
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  <li>Centrifuge the GeneJET spin column for 1 minute at 13,000 rpm
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  <li>Discard the flow through
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  <li>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li>Add 500 µl of wash solution to the column
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Discard flow through
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  <li>Centrifuge for 1 minute at 13,000 rpm
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  <li>Transfer the GeneJET column to a new 1.5 ml microcentrifuge tube
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  <li>Add 35 µl of ultrapure water. Do not touch the membrane with the pipette
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  <li>Incubate at room temperature for 2 minutes
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  <li>Centrifuge for 2 minutes at 13,000 rpm
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<p id="glycerolstock"><font color="#558e2b"><i>Glycerol cell stock generation</i></font></p>
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<p>This protocol is adapted from 2 freely available protocols<sup>REFERENCE, REFERENCE</sup>. Ignore steps 2-4 if antibiotic was not present in the overnight broth. Work in a sterile cabinet
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  <li>Take 0.5 ml from overnight culture and transfer to a centrifuge using sterile DNAase/RNAase free tips
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  <li>Centrifuge at 13,000 rpm for 2 minutes
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  <li>Discard supernatant
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  <li>Add 0.5 ml of 60% glycerol stock
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    <li>240 ml of glycerol
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    <li>160 ml nano pure water
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    <li>mix together and autoclave
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  <li>freeze at -80℃
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<!-- E. coli transformation -->
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<p id="ecolitransformation"><font color="#558e2b"><i>E. coli transformation</i></font></p>
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Another potential protocol
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<!-- Synechocystis transformation -->
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<p id="cyanotransformation"><font color="#558e2b"><i>Synechocystis transformation</i></font></p>
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Another potential protocol
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<td colspan="3"><h3 id="references"><font color="#558e2b">References</font></h3>
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1. http://www.thermoscientificbio.com/uploadedFiles/Resources/k070-product-information.pdf
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<td colspan="3"><h3 id="acknowledge"><font color="#558e2b">Acknowledgements</font></h3>
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Everyone we need to thank for help with protocols.
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Revision as of 20:26, 14 October 2014

University of Reading
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