Team:University of Chicago/Notebook/DH5-alpha competent cells

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(Difference between revisions)
(New page: ==Materials== ===TB Solution=== *10mM PIPES *15mM CaCl2 *250mM KCl *Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add 55mM M...)
(→Procedure)
 
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==Procedure==
==Procedure==
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1. Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.
+
# Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.
-
2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml
+
# Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml
overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an  
overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an  
A600 of 0.4-0.6.
A600 of 0.4-0.6.
*Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.
*Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.
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3. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
+
#Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
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4. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
+
# Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
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5. Spin cells 3K, 10', 4°C.
+
# Spin cells 3K, 10', 4°C.
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6. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
+
#Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
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7. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and  
+
# Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and  
freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106
freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106
cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.
cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.

Latest revision as of 16:18, 7 August 2008

Contents

Materials

TB Solution

  • 10mM PIPES
  • 15mM CaCl2
  • 250mM KCl
  • Dissolve in nanopure water and adjust pH 6.7 with KOH or HCL (solutes will dissolve as you do this) and then add

55mM MnCl2. Adjust to final volume. Sterilize by filtration with 0.45um filter and store at 4°C

LB Media

(per 1L batch)

  • 30 g LB mix
  • Dissolve in ddH2O, filling to 1L.
  • for 20mM MgSO4 FIRST add 16mL 1M MgSO4 solution THEN fill to 1L


Procedure

  1. Inoculate a 3ml overnight of E.coli in LB+20 mM MgSO4.
  2. Next morning, inoculate 250 ml LB+20 mM Mg++ in a 2L flask with about 2ml

overnight culture. Grow at room temp (23°C) with good aeration (250rpm) to an A600 of 0.4-0.6.

  • Note: TEMPERATURE IS IMPORTANT! At 37C cells will grow up to proper OD in ~3 hours. A faster growing time, however, compromises efficiency, so choose temperature accordingly.
  1. Place cells 10 min on ice. Transfer to a sterile bottle and spin 3K, 10', 4°C.
  2. Resuspend pellet in 80 ml cold TB (swirl cells in bottle). Leave 10’/ice.
  3. Spin cells 3K, 10', 4°C.
  4. Resuspend cells in 20 ml cold TB then add 1.5 ml DMSO. Leave 10'/ice.
  5. Dispense into 220 ul and 525 ul aliquots (in cold sterile tubes) and

freeze in dry ice/EtOH bath. Store -70°C. Typically, competency about 5X 106 cfu/ug DNA. Note, improves after freezing. Cells good for a year and counting.

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