Difference between revisions of "Team:Kent/Experiments"
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<a href="#Miniprep">Miniprep</a> </h3> | <a href="#Miniprep">Miniprep</a> </h3> | ||
− | <a name="Competent Cells"></a><h5> Competent Cells </h5> | + | <a name="Competent Cells"></a><b><h5> Competent Cells </b> </h5> |
<a href="#Overview">Overview</a> <br> | <a href="#Overview">Overview</a> <br> | ||
<a href="#Materials">Materials</a> <br> | <a href="#Materials">Materials</a> <br> |
Revision as of 11:48, 30 June 2015
Experiments & Protocols
Contents
Competent Cells
Transformation Protocol
Miniprep
Competent Cells
OverviewMaterials
References
Transformation Protocol
OverviewMaterials
Procedure
References
Miniprep
Overview
Procedure
- Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer it to a microcentrifuge tube.
- 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. If using LyseBlue reagent, the solution will turn blue.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
- Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through
- Centrifuge for 1 min to remove residual wash buffer.
- Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
- Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.