Topo Zero Blunt Vector by Promega Amplification of insert with M13 - Amplification of insert with M13 primers (Jun/07/2006 )
I have this insert in a Topo vector that supposedly has M13 sequences flanking it. However, I have tried to amplify the insert using M13 PRIMERS (-27) & (48), (-27) & (41), (-20) & (48). I have tried and it just doesnt seem to amplify. Instead all I see are primer dimers around the hundred region. I know the insert is there. Anyone has any idea what is going on.
Problem come from the PCR.
You may have to adjust the MgCl2 concentration. Using M13 primers, we use per 50µl reaction 0,5 µl of DMSO and MPgCl2 (50mM) is added as follows :
0µl if total extension is <2000pb (consider your fragment and vector amplif)
1,5µl for total amplif up to 3800
3µl for amplif > of 3800
Do you have a positive amplification control?