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Team:UNC Chapel Hill/4 June 2009
From 2009.igem.org
This what we did in Scott's lab on 6/4/09:
Contents |
Goals of Lab
- Learn how to do basic DNA transformation of E. Coli using plasmids
- Trying using the iGem parts, by putting Part BBa_I13521 into E. Coli [http://partsregistry.org/Part:BBa_I13521]
Terminology
Chemically competent E. Coli Electrocompetent E. Coli Electroporation Plasmid Ampicillin Heat Shock
Gist of Lab
Have six different groups:
1 - Chemically Competent E. Coli, with iGem BBa_I13521 Plasmid (Experimental group) 2 - Chemically Competent E. Coli, with pBluescript Plasmid (Positive Control) 3 - Chemically Competent E. Coli with no plasmid (Negative Control) 4 - Electrocompetent E. Coli with iGem BBa_I13521 Plasmid (Experimental group) 5 - Electrocompetent E. Coli with pBluescript Plasmid (Positive Control) 6 - Electrocompetent E. Coli with no plasmid (Negative Control)
pBluescript is a plasmid that Scott has that is known to work and make the cells white in Bluewhite screening [http://en.wikipedia.org/wiki/PBluescript]. Therefore 2 and 5 are our positive controls. 3 and 6 have no plasmids and are our negative controls. 1 and 4 are our experimental groups because we want to try see what happens by transforming the iGem plasmid using the same procedure.
Procedure
Notes:
- Keep Microcentrifuge tubes on ice unless indicated otherwise!
1.) Find the iGem part of interest. Puncture the appropriate well and inject 15 µL of dH20. Let sit for a couple of minutes to homogenize. The liquid will turn red due to an indicator in the well. 2.) Label 6 Microcentrifuge Tubes numbers 1-6 and sit them in ice. It's important for them to stayed cold, so that when the bacteria is transferred, the bacteria will not be heat shocked or damaged. 3.) Find tubes for bacteria of interest from the freezer, in this case:
- DH5α-E Chemically competent bacteria (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html)
- Electocompetent bacteria
4.) Let the bacteria thaw in the ice until a slurry. Place 25 µL of bacteria into the appropriate tube (CC in 1 through 3; EC in 4 through 6) 5.) Inject 1 µL of iGem part liquid into the appropriate tubes (1 and 4). Likewise for the pBluescript (2 and 5) 6.) Let the CC cells sit for 10 minutes. Afterward, heat shock the tubes at 40 degrees Celsius for 30 seconds each. Then add 500 µL of broth to each tube and put back onto ice. 7.) Electroporate 4-6 at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice. Immediately add 500 µL of broth after shocking and place back onto ice. 8.) Incubate all the tubes in a shaker for about 1 hour at 37 degrees Celsius. 9.) Plate 50 µL of bacteria on a corresponding agar dish with ampicillin. 10.) Incubate overnight in the oven. Check the colonies the next day.
