Drummond:Competent Cells: Difference between revisions

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(New page: 1) Take a 100uL aliquot of frozen cells from the -80C and inoculate a 3mL over night culture in LB. Do not add antibiotic. Work as sterile as possible. Grow this tube at 37C. 2) Take a...)
 
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1) Take a 100uL aliquot of frozen cells from the -80C and inoculate a 3mL over night culture in LB.  Do not add antibiotic.  Work as sterile as possible.  Grow this tube at 37C.
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2) Take a 1mL of overnight culture and inoculate 500mL LB broth (or 2mL into 1 L). 
==Materials==
We use cells with the following genotype: Tetr, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44,  thi-1, recA1, gyrA96, relA1, lac Hte, [ F', proAB, lacIqZΔM15 ,Tn10(tetr) Amy Camr]a


3) Grow this flask at 37C for 3.5-4 hours until an OD of 0.3-0.4 is reached.  If the OD is too low (0.1-0.2), its OK, the cells will still become competent.
* 1L Luria-Bertani (LB) broth
* XmL CaCl<sub>2</sub>
* XmL MgCl<sub>2</sub>


4) Centrifuge these cells at 5,000 for 10 minutes at 4C.  It may be easier to divide your cells into 2 250mL batches, but it is not necessary.  Keep the cells cold.  While spinning, ice down 100mM  CaCl2 and 100mM MgCl2 solutions.
==Protocol==


5) Gently re-suspend the bacterial pellet in 1/4 volume of ice cold MgCl2, taking 3-5 minutes for this procedure.   
# Inoculate a 3mL overnight culture of <i>E. coli</i> cells (our genotype below) in LB at 37°C.  Do not add antibiotic.  Work as sterile as possible.
# Take 1mL of overnight culture and inoculate 500mL LB broth (or 2mL into 1L). 
# Grow this flask at 37°C for 3.5-4 hours until an OD600 of 0.3-0.4 is reached.  Higher ODs will yield cells with impaired competence; lower ODs will result in fewer cells.
# Centrifuge these cells at 5,000g for 10 minutes at 4°C.  It may be easier to divide your cells into two 250mL batches, but it is not necessary.  Keep cells on ice.  While spinning, ice down 100mM CaCl<sub>2</sub> and 100mM MgCl<sub>2</sub> solutions.
# Gently resuspend the bacterial pellet in 1/4 volume of ice-cold MgCl<sub>2</sub>, taking 3-5 minutes for this procedure.   
# Centrifuge the cell suspension at 4,000g at 4°C for ten minutes.  Discard the supernatant.
# Resuspend the bacterial pellet on ice in 1/20 volume of ice cold CaCl<sub>2</sub> and then add an additional 9/20 volume of ice cold CaCl<sub>2</sub>.  Keep this suspension on ice for 20 minutes.
# Centrifuge the cell suspension at 4,000g at 4°C for 10 minutes. Discard the supernatant.
# Resuspend the cell pellet in 1/50 volume of ice-cold sterile 85mM CaCl<sub>2</sub> in 15% glycerol w/v.
# Dispense in 100μL aliquots and freeze at -80°C.


6) Centrifuge the cell suspension at 4,000RPM at 4C for ten minutes.
1L of culture yields X aliquots.


7) Re-suspend the bacterial pellet on ice in 1/20 volume of ice cold CaCl2 and then add an additional 9/20 volume of ice cold CaCl2.  Keep this suspension on ice for 20 minutes.
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8) Centrifuge the cell suspension at 4,000RPM at 4C for 10 minutes.
 
9) Re-suspend the cell pellet in 1/50 volume of ice cold sterile 85mM CaCl2 in 15%glycerol w/v.
 
10) Dispense in 100uL aliquots and freeze at -80C.

Revision as of 19:02, 2 April 2009

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Materials

We use cells with the following genotype: Tetr, Δ(mcrA)183, Δ(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac Hte, [ F', proAB, lacIqZΔM15 ,Tn10(tetr) Amy Camr]a

  • 1L Luria-Bertani (LB) broth
  • XmL CaCl2
  • XmL MgCl2

Protocol

  1. Inoculate a 3mL overnight culture of E. coli cells (our genotype below) in LB at 37°C. Do not add antibiotic. Work as sterile as possible.
  2. Take 1mL of overnight culture and inoculate 500mL LB broth (or 2mL into 1L).
  3. Grow this flask at 37°C for 3.5-4 hours until an OD600 of 0.3-0.4 is reached. Higher ODs will yield cells with impaired competence; lower ODs will result in fewer cells.
  4. Centrifuge these cells at 5,000g for 10 minutes at 4°C. It may be easier to divide your cells into two 250mL batches, but it is not necessary. Keep cells on ice. While spinning, ice down 100mM CaCl2 and 100mM MgCl2 solutions.
  5. Gently resuspend the bacterial pellet in 1/4 volume of ice-cold MgCl2, taking 3-5 minutes for this procedure.
  6. Centrifuge the cell suspension at 4,000g at 4°C for ten minutes. Discard the supernatant.
  7. Resuspend the bacterial pellet on ice in 1/20 volume of ice cold CaCl2 and then add an additional 9/20 volume of ice cold CaCl2. Keep this suspension on ice for 20 minutes.
  8. Centrifuge the cell suspension at 4,000g at 4°C for 10 minutes. Discard the supernatant.
  9. Resuspend the cell pellet in 1/50 volume of ice-cold sterile 85mM CaCl2 in 15% glycerol w/v.
  10. Dispense in 100μL aliquots and freeze at -80°C.

1L of culture yields X aliquots.

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