Team:University of Lethbridge/Notebook/Project1August

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(Difference between revisions)
m (August 13, 14)
m (→Selina, Munima)
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'''Preparation of Chemically Competent Cells in CaCl2'''
'''Preparation of Chemically Competent Cells in CaCl2'''
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Protocol (adapted based on materials availability from Sambrook & Russell, (2001) ''Molecular Cloning: A Laboratory Manual 3rd Edition. Protocol 25'' '''Vol. 1; 1.117'''):
+
Protocol (adapted 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.
+
  1. Inoculated 100 mL liquid LB media using 100 uL of glycerol stocked RP1616 cells from iGEM07. Incubated at 37C
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  2. Transfer cells to sterile, ice-cold 35 mL centrifuge tube (NOT FALCON TUBE). Cool culture by incubating on ice for 10 minutes.
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      for 4 hours. Measured A600 to be 0.666 (ideal A600 = 0.4 - 0.6).
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  3. Pellet cells by centrifugation at 2000 rpm for 5 min and decant supernatant.
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  2. Transferred cells to sterile, ice-cold 35 mL centrifuge tube (NOT FALCON TUBE). Cooled culture on ice for  
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  4. Resuspend, gently, using Pasteur pipette, in ___ mL of ice-cold 80 mM MgGl2-20 mM CaCl2 solution.
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      10 minutes.
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  5. Recover cells by centrifugation at 2500 rpm for 5 min and decant supernatant.
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  3. Pelleted cells by centrifugation at 2000 rpm for 5 min and decanted supernatant.
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  6. Resuspend cells, by gentle swirling, in 2 mL of 100 mL CaCl2 for each 30 mL of original culture.
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  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.
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Added 300 uL of cells into pre-aliquoted glycerol stock tubes. Made 1/2? glycerol stocks of RP1616 chemically competent cells. ???
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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'''
'''Transformation of CaCl2 Competent Cells with Plasmid DNA'''
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Did a dilution series of plasmid DNA in competent RP1616 cells.  
+
Attempted to transform pTopp and pSB1A7 (positive control) into (hopefully) CaCl2 competent RP1616 cells.
Protocol:
Protocol:
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  1. Into 200 uL of chem. competent cells, add pTopp (5, 10 or 20 uL) pSB1A7 (5, 10 uL).
+
  1. Added plasmid solutions pTopp (5, 10 or 20 uL) or pSB1A7 (5, 10 uL)into 200 uL of chem. competent cells.
-
  2. Incubate cells on ice for 30 minutes.
+
      -pTopp plasmid prep from ______, pSB1A7 from ______
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  3. Heat shock cells for 2 minutes at 42 C (for 1.5 mL microcentrifuge tube).
+
  2. Incubated cells on ice for 30 minutes.
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  4. Incubate on ice for 5 minutes.
+
  3. Heat shocked cells for 2 minutes at 42 C (for 1.5 mL microcentrifuge tube).
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  5. Add 1.0 mL of pre-warmed LB media.
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  4. Incubated on ice for 5 minutes.
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  6. Incubate at 37 C for 60 minutes with gentle shaking (~100 rpm).
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  5. Added 1.0 mL of pre-warmed LB media.
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  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.
+
  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.
  8. Spread the 300 uL suspension on an LB + Amp plate.
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  9. Incubate overnight at 37 C.
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  9. Incubated overnight at 37 C.
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===August 14, 2008===
===August 14, 2008===
====Selina====
====Selina====
No growth on any of the plates.
No growth on any of the plates.

Revision as of 02:05, 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 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

No growth on any of the plates.

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