Maloof Lab:Cell Transformation: Difference between revisions
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<h3><font style="color:#F8B603;"> | <h3><font style="color:#F8B603;">E. coli (DH5α chemically-competent) Transformation</font></h3> | ||
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#Thaw one aliquot of DH5α competent cells (from –80°C freezer) on ice | #Thaw one aliquot of DH5α competent cells (from –80°C freezer) on ice | ||
#Add 5 μl DNA and | #Add 1-5 μl DNA and 75 μl competent cells to new cell culture tube. <b>Do not mix the cells by pipetting</b>. Just tap the tube gently | ||
#Incubate on ice for 30 minutes | #Incubate on ice for 30 minutes | ||
#Heat shock in 42°C water bath for 30 seconds | #Heat shock in 42°C water bath for exactly 30 seconds. Do not mix or shake | ||
#Cool on ice | #Cool on ice | ||
#Add 900 μl liquid media (e.g. LB) to cell culture tube | #Add 900 μl liquid media (e.g. LB) to cell culture tube | ||
#Shake at 37°C for 40-60 minutes | #Shake at 37°C for 40-60 minutes | ||
#Centrifuge at 5000 rpm for | #Centrifuge at 5000 rpm for 2 minutes | ||
#Remove all but 100-150 μl of the LB and resuspend pellet | #Remove all but 100-150 μl of the LB and resuspend pellet | ||
#Spread 100-150 μl on plate of media + antibiotic (e.g. LB agar + carb) | #Spread 100-150 μl on plate of media + antibiotic (e.g. LB agar + carb) | ||
Latest revision as of 17:29, 29 May 2009
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Room 2115 |
E. coli (DH5α chemically-competent) Transformation
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