"
Team:University of Lethbridge/Notebook/Project1August
From 2008.igem.org
Contents |
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.
___
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.
August 15, 2008
Munima
Objective: Prepare cells for electroporation/chemical competency.
Streaked RP1616 (from glycerol stock) onto LB plate and inoculated 5 mL LB liquid media tube with the same culture. Streaked glycerol stocked pSB1A7 onto LB + Amp plate - will plasmid prep to replenish pSB1A7 stock in the -20 C freezer.
August 16, 2008
Selina
Objective: Reattempt to create CaCl2 chemically competent RP1616 E. coli cells, following Sambrook's protocol instead of using modified version from Mosimann lab
Modifications to Aug. 13 protocol:
-Used cell culture of OD600 = 0.503 -Final resuspension of 1 mL CaCl2 per ~35 mL cells
Streaked CaCl2 treated cells on LB plate directly after competency treatment
Created glycerol stocks labeled 'CaCl2 treated RP1616' and placed in HJ's -80C
Used CaCl2 cells for Sambrook's direct-from-CaCl2-treatment transformation protocol.
Volumes used for transformations:
-100 mL CaCl2 cell suspension per transformation
-Varying amounts of plasmid solution (Sambrook advises <5% of cell culture volume and max 25ng/50mL cell culture
-pTopp: 1 uL, 2.5 uL, 5 uL
-pSB1A7: 1 uL, 2.5 uL, 5 uL
-pUC19 (positive control): 1 uL, 2.5 uL
-Plated resulting cells on LB + Amp plates and left overnight at 37C
