Team:EPF-Lausanne/Protocols/Competent cells. Protocol II

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==Uses==
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{{back to protocols}}
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TB buffer is used to make chemically competent cells by the Inoue method
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==Materials==
==Materials==
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* PIPES buffer 0.5M solution pH 6.7  (sterilized, made with milli-Q pure H2O, store at -20C)
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* Plate of cells streaked for single colonies
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* Manganese chloride tetrahydrate
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* [[SOB]]
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* Calcium chloride dihydrate
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* Ice
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* Potassium chloride
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* [[TB buffer]]
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* Potassium hydroxide 1M solution
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* DMSO
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* Sterile 0.22 μm bottle top filter
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* Dry Ice (or liquid nitrogen)
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* vacuum source
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* clean glassware (no detergent residue -- clean with 70% EtOH)
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==Glassware & equipment==
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* 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
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* 220 ml conical centrifuge tubes BD 35 2075
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* Eppendorf 5410R refrigerated centrifuge with conical adapters
==Preparation==
==Preparation==
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For a 1 liter solution add:
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# Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask.  Save some medium as an OD blank.
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* 250 mM potassium chloride (18.65 g)
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# Grow to  an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
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* 15 mM calcium chloride (2.2 g)
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# Prechill the centrifuge to 4 degrees
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* 10 mM PIPES (20 ml of an 0.5 M solution)
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# Remove from the incubator and place on ice for 10 minutes
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# Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
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# Drain the medium and resuspend each pellet first in 5 ml of ice cold TB.  Add  an additional 75 ml of cold TB buffer and resuspend.
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# Place on ice for 10 minutes
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# Spin down as above.
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# While spinning, add 1.4 ml of DMSO to 18.6 ml of TB  (7% DMSO mixture)
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# Resuspend each pellet in 20 ml of cold TB-DMSO mixture
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# Incubate on ice for 10 minutes
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# Dispense cells into pre-chilled tubes
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# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely
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to 800 ml of DI water.  Adjust pH to 6.7 with 1M KOH.
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==Thoughts on improvements==
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* "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
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* They also control pH at 7.5, which may be a major issue
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* Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
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* Length of time on ice prior to transformation may make a big difference
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* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference.  Let's see.
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* Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.
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* My lab uses LB for instead of SOB media...it seems to work fine for them--[[User:Melissali|mel]] 18:10, 14 June 2007 (EDT)
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Dissolve
 
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* 55 mM manganese chloride (10.88 g)
 
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in 100 ml of DI water, add gradually to the solution of the remaining components.  Bring solution to 1 liter.  The pH of the solution will fall, which is expected.
 
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* Do not attempt to adjust the pH.  Adding base after adding manganese will precipitate a yellow/brown hydroxide.
 
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Sterile filter with a pre-washed 0.22 μm filter and store indefinitely at 4 degrees.
 
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Revision as of 09:44, 8 July 2011

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