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Revision as of 10:32, 12 July 2013 (view source) FrederikM (Talk | contribs) ← Older edit		Revision as of 10:35, 12 July 2013 (view source) FrederikM (Talk | contribs) (→Chemically competent E.coli cells: Short CaCl2 method) Newer edit →
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-	== Chemically competent E.coli cells: Short CaCl2 method ==	+	== Chemically competent E.coli cells: Short CaCl<math>_2</math> method ==
	=== Procedure ===		=== Procedure ===

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Revision as of 10:35, 12 July 2013

Contents 1 Chemically competent E.coli cells: Short CaCl<math>_2</math> method 1.1 Procedure 1.2 Buffers and solutions 2 Chemically competent E.coli cells: Inoue method 2.1 Procedure 2.2 Buffers and solutions

Chemically competent E.coli cells: Short CaCl<math>_2</math> 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