From 2012.igem.org
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| - | '''Transformation Protocol Using Heat Shock'''
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| - | 1) Take competent ''E.coli'' cells from –80°C freezer.
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| - | 2) Turn on water bath to 42°C.
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| - | 3) Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a
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| - | DNA construct, use 50 ul of competent cells. For transforming a ligation, use 100
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| - | ul of competent cells. You may need more or less cells, depending how competent
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| - | they are.
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| - | 4) Keep tubes on ice.
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| - | 5) Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 min. to thaw
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| - | competent cells.
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| - | 6) Put tube(s) with DNA and E.coli into water bath at 42°C for 45 seconds.
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| - | 7) Put tubes back on ice for 2 minutes to reduce damage to the ''E.coli'' cells.
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| - | 8) Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37°C.
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| - | (Can incubate tubes for 30 minutes, unless trying to grow DNA for ligation which
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| - | is more sensitive. For ligation, leave tubes for 1 hour).
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| - | 9) Spread about 100 ul of the resulting culture on LB plates (with appropriate
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| - | antibiotic added – usually Ampicillin or Kanamycin.) Grow overnight.
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| - | 10) Pick colonies about 12-16 hours later.
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Latest revision as of 07:51, 23 September 2012
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