Difference between revisions of "Team:Kent/Experiments"
Line 14: | Line 14: | ||
<a name="Competent Cells"></a><h5> Competent Cells</h5> | <a name="Competent Cells"></a><h5> Competent Cells</h5> | ||
<a href="#Overview">Overview</a> <br> | <a href="#Overview">Overview</a> <br> | ||
+ | <li>Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which foreign DNA can be passed through easily. </li> | ||
+ | <li> E. coli cells that have been specially treated to transform efficiently. </li> | ||
<a href="#Materials">Materials</a> <br> | <a href="#Materials">Materials</a> <br> | ||
<li>3ml 1M MnCl<sub>2</sub> </li> | <li>3ml 1M MnCl<sub>2</sub> </li> |
Revision as of 12:01, 30 June 2015
Experiments & Protocols
Contents
Competent Cells
Transformation Protocol
Miniprep
Competent Cells
Overview- Overnight culture of VS45 cells are back-diluted to OD600 0.1 in 250 ml LB broth
- The cells are then grown at 37˚C to OD600 0.6 and then harvested by centrifugation.
- The cells are resuspended in 100 ml of prechilled buffer and incubated on ice for 60 minutes.
- Harvest again by centrifugation (at 4˚C), and resuspended in 5 ml of pre-chilled buffer.
- The resuspended cells can then be aliquoted (on ice), frozen using dry ice or liquid nitrogen, and stored at -80˚C.
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.