PCR - (Feb/21/2007 )
If anybody can suggest me what is the best conditions for colony PCR: I have done cloning in pGEM Teasy vector
I always get nonspecific bands in addition to the specific band of my interest...also i get DNA band in my negative control where there z no DNA except the master mix
Pick a colony using a toothpick, touch on fresh LB plate and then insert it into PCR tube. Then you can run PCR with longer duration of denaturation step.
About the same, except I substitute tooth picks with yellow pipette tip (they make a nice sharp but blunt end) I place the tips into 50ul of sterile distilled water. And use 2.5ul of that solution for the PCR reaction, total volume 10ul.
It appears that PCR is sensitive enough to detect unligate DNA fragments sitting on the gel surface. So always PCR across juntions between DNA fragments, this avoid any false positives from the above source.
actually i wanted to know the conditions for denaturaion, annaeling and extension...if any body can suggest me reagrding these conditions...
This Colony PCR discussion should give you some idea of the conditions people use.
I think someone should pin that discussion up for easier viewing.