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Team:University of Lethbridge/Notebook/Project1August
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August 13, 2008
Selina, Munima
Objective: Second attempt at making chemically competent RP1616 cells and transforming them with pTopp and pSB1A7.
Preparation of Chemically Competent Cells in CaCl2
Protocol (adapted from Sambrook & Russell, (2001) Molecular Cloning: A Laboratory Manual 3rd Edition. Protocol 25 Vol. 1; 1.117):
1. Inoculated 100 mL liquid LB media using 100 uL of glycerol stocked RP1616 cells from iGEM07. Incubated at 37C
for 4 hours. Measured A600 to be 0.666 (ideal A600 = 0.4 - 0.6).
2. Transferred cells to sterile, ice-cold 35 mL centrifuge tube (NOT FALCON TUBE). Cooled culture on ice for
10 minutes.
3. Pelleted cells by centrifugation at 2000 rpm for 5 min and decanted supernatant.
4. Resuspended gently, using Pasteur pipette, in 20 mL of ice-cold 80 mM MgCl2-20 mM CaCl2 solution.
5. Centrifuged cells at 2500 rpm for 5 min and decanted supernatant.
6. Resuspended cells, by gentle swirling, in 2 mL of 100 mL CaCl2.
Created one 30 % glycerol stock and two (accidentally) 50% glycerol stocks. Placed temporarily in Steve's -80C freezer in Selina's box.
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Transformation of CaCl2 Competent Cells with Plasmid DNA
Attempted to transform pTopp and pSB1A7 (positive control) into (hopefully) CaCl2 competent RP1616 cells.
Protocol:
1. Added plasmid solutions pTopp (5, 10 or 20 uL) or pSB1A7 (5, 10 uL)into 200 uL of chem. competent cells.
-pTopp plasmid prep from ______, pSB1A7 from ______
2. Incubated cells on ice for 30 minutes.
3. Heat shocked cells for 2 minutes at 42 C (for 1.5 mL microcentrifuge tube).
4. Incubated on ice for 5 minutes.
5. Added 1.0 mL of pre-warmed LB media.
6. Incubated at 37 C for 60 minutes with gentle shaking (~100 rpm).
7. Pelleted cells at 6000g for 3 minutes (7000 rpm) and removed 700 uL. Resuspended the cell pellet in the remaining
300 uL by gentle pipetting.
8. Spread the 300 uL suspension on an LB + Amp plate.
9. Incubated overnight at 37 C.
August 14, 2008
Selina
Checked transformation plates (Aug. 12, RP1616 + pTopp or pSB1A7 plasmid) after 14.5 hours.
No growth on any of the plates.
