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Team:Stanford/Protocols
From 2010.igem.org
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| + | ==Digestion Recipe== | ||
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| + | ==Ligation Recipe== | ||
==Heat-Shock Transformation== | ==Heat-Shock Transformation== | ||
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#Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight. | #Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight. | ||
#Pick the colonies about 12-16 hours later. | #Pick the colonies about 12-16 hours later. | ||
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Revision as of 22:24, 21 July 2010
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Digestion Recipe
Ligation Recipe
Heat-Shock Transformation
Cells
- E. coli DH5alpha
Protocol
- Take out competent E.coli cells from –80 C freezer.
- Turn on water bath to 42 C.
- Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar) – you may need more or less cells, depending how competent they are.
- Keep tubes on ice.
- Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
- Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
- Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
- Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
- Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
- Pick the colonies about 12-16 hours later.
