Team:University of Lethbridge/Notebook/Project1August

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===August 13, 2008===
===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 based on materials availability from Sambrook & Russell, (2001) ''Molecular Cloning: A Laboratory Manual 3rd Edition. Protocol 25'' '''Vol. 1; 1.117'''):
 +
1. Inoculate 1 mL of glycerol stocked RP1616 cells from iGEM07 into 100 mL liquid LB media.  Shake at 37 C until A600 is 0.4.
 +
2. Transfer cells to sterile, ice-cold 35 mL centrifuge tube (NOT FALCON TUBE). Cool culture by incubating on ice for 10 minutes.
 +
3. Pellet cells by centrifugation at 2000 rpm for 5 min and decant supernatant.
 +
4. Resuspend, gently, using Pasteur pipette, in ___ mL of ice-cold 80 mM MgGl2-20 mM CaCl2 solution.
 +
5. Recover cells by centrifugation at 2500 rpm for 5 min and decant supernatant.
 +
6. Resuspend cells, by gentle swirling, in 2 mL of 100 mL CaCl2 for each 30 mL of original culture.
 +
 +
Added 300 uL of cells into pre-aliquoted glycerol stock tubes. Made 1/2? glycerol stocks of RP1616 chemically competent cells. ???
 +
 +
___
 +
 +
'''Transformation of CaCl2 Competent Cells with Plasmid DNA'''
 +
 +
Did a dilution series of plasmid DNA in competent RP1616 cells.
 +
 +
Protocol:
 +
1. Into 200 uL of chem. competent cells, add pTopp (5, 10 or 20 uL) pSB1A7 (5, 10 uL).
 +
2. Incubate cells on ice for 30 minutes.
 +
3. Heat shock cells for 2 minutes at 42 C (for 1.5 mL microcentrifuge tube).
 +
4. Incubate on ice for 5 minutes.
 +
5. Add 1.0 mL of pre-warmed LB media.
 +
6. Incubate at 37 C for 60 minutes with gentle shaking (~100 rpm).
 +
7. Pellet cells at 6000g for 3 minutes (____ rpm) and remove ~700 uL. Resuspend the cell pellet in the remaining 300 uL by gentle pipetting.
 +
8. Spread the 300 uL suspension on an LB + Amp plate.
 +
9. Incubate overnight at 37 C.
 +
 +
 +
===August 14, 2008===
 +
====Selina====
 +
No growth on any of the plates.

Revision as of 01:35, 15 August 2008

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 based on materials availability from Sambrook & Russell, (2001) Molecular Cloning: A Laboratory Manual 3rd Edition. Protocol 25 Vol. 1; 1.117): 

1. Inoculate 1 mL of glycerol stocked RP1616 cells from iGEM07 into 100 mL liquid LB media.  Shake at 37 C until A600 is 0.4.
2. Transfer cells to sterile, ice-cold 35 mL centrifuge tube (NOT FALCON TUBE). Cool culture by incubating on ice for 10 minutes.
3. Pellet cells by centrifugation at 2000 rpm for 5 min and decant supernatant.
4. Resuspend, gently, using Pasteur pipette, in ___ mL of ice-cold 80 mM MgGl2-20 mM CaCl2 solution.
5. Recover cells by centrifugation at 2500 rpm for 5 min and decant supernatant.
6. Resuspend cells, by gentle swirling, in 2 mL of 100 mL CaCl2 for each 30 mL of original culture.

Added 300 uL of cells into pre-aliquoted glycerol stock tubes. Made 1/2? glycerol stocks of RP1616 chemically competent cells. ???

___

Transformation of CaCl2 Competent Cells with Plasmid DNA

Did a dilution series of plasmid DNA in competent RP1616 cells.

Protocol: 

1. Into 200 uL of chem. competent cells, add pTopp (5, 10 or 20 uL) pSB1A7 (5, 10 uL).
2. Incubate cells on ice for 30 minutes.
3. Heat shock cells for 2 minutes at 42 C (for 1.5 mL microcentrifuge tube).
4. Incubate on ice for 5 minutes.
5. Add 1.0 mL of pre-warmed LB media.
6. Incubate at 37 C for 60 minutes with gentle shaking (~100 rpm).
7. Pellet cells at 6000g for 3 minutes (____ rpm) and remove ~700 uL. Resuspend the cell pellet in the remaining 300 uL by gentle pipetting.
8. Spread the 300 uL suspension on an LB + Amp plate.
9. Incubate overnight at 37 C.


August 14, 2008

Selina

No growth on any of the plates.

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