Team:KU Leuven/Protocols

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== Chemically competent E.coli cells: CaCl <html><math> _{2}</math></html> method ==
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== Chemically competent E.coli cells: CaCl <html><math> ^{2}</math></html> method ==
=== Procedure ===
=== Procedure ===

Revision as of 10:50, 12 July 2013

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Contents

Chemically competent E.coli cells: CaCl ^{2} method

Procedure

Perform every action on ice – also resuspending your cells !! Do not shock freeze (liqN2) – just transfer from ice to -80°C !!! Work sterile

  • Inoculate 3ml growth medium with your cells of choice (DH5alpha or TOP10 for plasmid maintenance & cloning)
  • Grow overnight at 37°C with sufficient aeration
  • Inoculate 100 ml LB with 1 ml of overnight culture
  • Grow at 37°C to an Optical Density600nm of approx 0.5 to 0.8 (usually 2-3 hrs)
  • Centrifuge cells (3700-4000 rpm 4°C 12min – sterile 50ml tube)
  • Re-suspend pellet on ice with FSB to 15 ml (cold) for each 100 ml pellet
  • Incubate cells 10min on ice
  • Centrifuge cells (3700 – 4000 rpm 4°C 10min)
  • Re-suspend pellet on ice in 4-8 ml FSB (cold) for each 100 ml pellet
  • Aliquot cells appropriately (200-400 µl aliquots) and freeze aliquots at -80°C

Buffers and solutions

  1. Growth medium
    • LB 25g/l
  1. FSB
    • 10 mM Potassium Acetate
    • 10% glycerol
    • 10 mMKCl
    • 50 mM CaCl2
    • Check pH – must be around 6.2 – if need be adjust with AcAc (HCl) or KOH
    • Buffer should be filter-sterilized (0.45 micrometer filter) but has been autoclaved also

Chemically competent E.coli cells: Inoue method

Procedure

Perform every action on ice – also resuspending your cells !! Work sterile

  • Pick a single colony from a freshly transformed plate (after overnight growth @ 37 degrees)
  • Transfer the colony to 25 ml growth medium in a 250 ml erlenmeyer (sterile !)
  • Incubate the culture @ 37°C for 6 – 8 hrs under vigourous shaking (250 – 300 rpm)
  • Prepare 3 1L flasks with 250 ml growth medium in each
  • Inoculate the flasks with 10, 4 or 2 ml of the dayculture -> you create 3 different starting optical densities.
  • Incubate the cultures @ 18-22°C overnight under moderate shaking (180 – 220 rpm)
  • Monitor the OD600nm until it reaches 0.55
  • Place cells in an ice-water bath to cool them down quickly (-> swirl occasionally, keep them in for approx 10min)
  • Harvest cells @4°C for 10min at 2500g
  • Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
  • Resuspend gently (swirl !) in 80 ml icecoldinoue transformation buffer
  • Harvest cells @4°C for 10min at 2500g
  • Pour off supernatant (to biological waste) – make sure all remaining droplets are removed
  • Resuspend gently (swirl !) in 20 ml icecoldinoue transformation buffer
  • Add 1.5 ml 100% DMSO – mix by swirling
  • Store whole on ice for approx 10 minutes
  • Aliquot as quickly as possible 100 – 200 microliter aliquots into 1.5 ml tubes (precooled on ice) and snapfreeze them into a liqN2 bath

Buffers and solutions

  1. Growth medium
  2. Inoue transformation buffer
Reagent Final concentration (mM) Amount per liter
MnCl2 55 10.88g (from MnCl2*4H2O)
CaCl2 15 2.20g (from CaCl2*2H2O)
KCl 250 18.65g (from KCl)
PIPES 10 20ml (from 0.5M stock solution)
H2O to 1 liter

Filter sterilize with a 0.45 micrometernalgene filter

  1. Stock 0.5M PIPES (piperazine-1,2-bis[2-ethanesulfonic acid]) pH 6.7
    • Dissolve 15.1g PIPES in 80ml MilliQ H2O
    • Adjust pH to 6.7 with 5M KOH
    • Bring volume to 100 ml with MilliQ H2O
    • Filter sterilize with a 0.45 micrometernalgene filter
    • Aliquot (5 times) and store at -20°C
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