In situ PCR of bacterial colonies?
Tue Nov 26 22:32:32 EST 1996
Hi! You want to PCR screen your transformants for the insert?
Cocktail for 21 colonies:
21 ul 10x PCR buffer
4.2 ul 10 mM dNTPs
2.1 ul of each 100 uM primer (forward and reverse sequencing primers for your
0.84 ul of 5U/ul Taq DNA polymerase
*bring to 210 ul final volume with sterile distilled water
*aliquot 10 ul of this cocktail to your PCR tubes
Pick your colony with a sterile toothpick, then dip it into
a 10 ul PCR reaction cocktail, then streak it onto an agar
plate. cover the PCR reactions with a drop of mineral oil.
I use the following PCR cycle for a 1.5 kb insert in pBluescript:
2 minutes @ 94 C
30x: 1 min. @ 92 C
1 min. @ 53 C
1.5 min. @ 72 C
10 min. @ 74 C
Run the entire PCR reaction on an agarose gel.
c/o Dr. G. Drouin
Dept of Biology
U of Ottawa
banoo at bio.uottawa.ca
goldberg at bms.com (Steven Goldberg) wrote:
>I was just wondering if anyone has developed or heard about an in situ PCR
>protocol for E. coli colonies carrying cloned genes. If you have any
>information on this topic I would like to hear from you.
More information about the Methods