Difference between revisions of "Team:Warwick/Protocols"

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             <h2>Transformation</h2>
 
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               <p>1.Collect electrocompetent cells from – 80 freezer and keep on ice.<br />2.Add 1 μl of plasmid to tube containing 55 μl of competent cells.<br />3.Place electroporation cuvettes on ice to chill.<br />4.Mix 56 μl mixture and transfer to chilled electroporation cuvettes, ensuring mixture is released between the two metal plates.<br />5.Ready 950 μl of SOC in a pipette.<br />6.Place the electroporation cuvette into the electroporator.<br />7.Make sure the electroporator is set to bacteria.<br />8.Slide electroporation cuvette into position.<br />9.Press pulse.<br />10.Immediately remove cuvette and lid, and add the 950 μl SOC.<br />11.Place cuvette on ice.<br />12.Transfer contents of cuvette back into Eppendorf (using 950 pipette) and label with the name of the plasmid.<br />13.Repeat for however many plasmids you wish to transform.<br />14.Allow cells to recover for 1 hour at 37 °C with the SOC.<br />15.Allow cells to grow on plates with appropriate antibiotic(s).</p>
 
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Revision as of 12:02, 18 October 2016

iGEM Warwick 2016 - Protocols

Using iGEM Distribution Kit

Bunsen required for all steps.
1.Label tube with the name of the plasmid being added.
2.Locate DNA on kit plate (see iGEM distribution catalog).
3.Pierce the foil with a pipette containing 10 μl of sterilized water.
4.Mix well by taking up and releasing mixture multiple times.
5.Place the suspended DNA solution in a 1.5 ml Eppendorf, leaving on ice.

Transformation

1.Collect electrocompetent cells from – 80 freezer and keep on ice.
2.Add 1 μl of plasmid to tube containing 55 μl of competent cells.
3.Place electroporation cuvettes on ice to chill.
4.Mix 56 μl mixture and transfer to chilled electroporation cuvettes, ensuring mixture is released between the two metal plates.
5.Ready 950 μl of SOC in a pipette.
6.Place the electroporation cuvette into the electroporator.
7.Make sure the electroporator is set to bacteria.
8.Slide electroporation cuvette into position.
9.Press pulse.
10.Immediately remove cuvette and lid, and add the 950 μl SOC.
11.Place cuvette on ice.
12.Transfer contents of cuvette back into Eppendorf (using 950 pipette) and label with the name of the plasmid.
13.Repeat for however many plasmids you wish to transform.
14.Allow cells to recover for 1 hour at 37 °C with the SOC.
15.Allow cells to grow on plates with appropriate antibiotic(s).