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Team:St Pauls London/Protocols
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Revision as of 11:52, 7 June 2013
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Protocols
All protocols were carried out following safety proceedures as detailed on the safety page. The methods used were commercially approved as mentioned below.
QIAGEN approved Plasmid DNA Extraction (a.k.a. The Miniprep):
All buffers used in this method were Biorad-supplied.
1) Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature (15-25°C).
2) Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3) Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
4) Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
5) Centrifuge for 10 min at 13,000 rpm in a table top microcentrifuge.
6) Apply the supernatant from Step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.
7) Was the QIAprep spin column by adding 500 μl Buffer PB. Centrifuge for 30-60s and discard the flow through.
8) Wash the QIAprep spin column to the collection tube.
9) Centrifuge for 1 min to remove residual wash buffer.
10) Place the QIAprep column in a clean 1.5 microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (or distilled water) to the centre of the QIAprep spin column, let it stand for 1 min, then centrifuge for 1 min.
NEB approved Transformation (High Efficiency)for 10-beta Competent E.Coli:
