PCR screeing for positive transormation - (May/04/2005 )

Does anyone have used PCR screening for positive transformation?
And, what condition will you suggest ?

~~ thank you

Hi Wenni,

This topic was recently discussed try:

http://www.protocol-online.org/forums/inde...t=ST&f=2&t=6235


However, I commonly use bacteria colony PCR for screening so if you just want a method.

I innoculate a PCR mix of water, 10 x buffer and dNTPs with a bacterial colony (don't forget to also innoculate some broth so you don't lose the colony). Heat this PCR mix for 10 minutes at 100 degrees celcius and allow to cool to room temp (not everyone does this but I find it helps). Add primers and Taq and do a 30 cycle PCR.

The best way is to use a primer within the vector (I commonly use M13F) and an internal fwd/rev primer. If this is not practical you can use your specific PCR primers for your insert.

Hope this helps

Cheers,

Scott