In situ PCR of bacterial colonies?

Shehrebanoo Malik banoo
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
vector)
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.


Good luck!


 Banoo Malik
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.
>
>Thanks.
>
>Steve




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