colony PCR

[Analia Alet]

Hi Sunil
You can find a good method for colony PCR at Sambrook et al. (Manniatis)
Also what I used to do was to start the PCR without Taq. After a first cycle 
of 5 min 91ºC, I stopped it manually and added Taq. Then I continued the PCR 
as usual.
This first cycle is important to disrupt the cells. Another advice is to put 
very but very little bacterial colony into the PCR tube. Just touch the 
colony, don't worry if you don't see the bacterias at the end of the tip 
(maybe that's the problem; perhaps you are placing half the entire colony into 
the PCR tube)

Finally, my PCR protocol (but it depends on your primers)

1 cycle 91ºC 5 min x1
stop manually, place in ice and add Taq
2 cycle (x35) 94ºC 1 min; 60ºC 1 min; 72ºC 1 min
3 cycle 72ºC 5 min x1
4 hold 4ºC

Hope it works for you
best regards

Analía


>I am in need of colony PCR method . I got the positive colonies for Agro but
>not getting the band for insert. I got the band for positive control in the
>same reaction.
>where would be the problem