no band obtain after PCR - (Aug/10/2005 )

dear all..

i got stucked into this problem..i got colony after transformation and extract the plasmid out..after digestion with the same restriction enzyme (that i used to cut vector and insert),i got the right size of band..and Thank God for that!

but unfortunately ,when i do the PCR to confirm whether the insert has been ligated or not, i didnt get any band at all..for the positive control (plasmid that has the insert),there were two bands..

why was that happen?i try to optimize the PCR mixture and annealing tempreture(range from 55.5-63) and still got no result from that..

help..

Did you only get one colony? Also your post is a little difficult to follow- did the correct colony give you no band or two bands?

Daniel

DNA sequencing reagents

Hi,
I was just wondering WHAT DO YOU TRANSFORM??

I mean..did u try to do the PCR reaction on the plasmid itself ( before transforming)??

you could even check with another restrictyion digestion to be completely sure..

good luck