Trouble with PCR using ligation mix as template? - (Mar/06/2015 )
I have successfully ligated two DNA fragments (2.5 kb and 4.2 kb), and used the ligation mix as a template for PCR to amplify the long fragment (6.7 kb). I also amplified both constituent fragments as positive controls. None of the PCRs worked - no bands apart from primers.
I know that the ligation worked because I ran some of it out on a gel. I tried cleaning up the ligation mix (Sigma kit) but it made no difference. The PCR protocol seems to be fine as the same PCRs worked fine on genomic DNA.
Any suggestions/advice would be greatly appreciated.
Details:
PCR: 2 uL template DNA, 2-2 uL primers (0.25mM), 25 uL RedTaq mix, 19 uL dH2O
95C 5 min
--cycle repeated 29 times:--
95C 30 s
55C 30 s
72C 2.5 min
------------------------------
72C 10 min
Most common error is far too much DNA template. Try again diluting your DNA 100x to 1000x.
Thanks, I'm running another few runs today using diluted template.
Forget the ligation step.
Amplify the 2.5 and 4.6kb fragments using primers with complementary ends. Gel purify the two and fuse the two with overlap extension pcr.
Some hints for difficult overlap extension:
Use DMSO (2-5%)
For the second round of overlap extension, use touchdown PCR , and use slow ramping rates. The PCR ends up taking 4-5 hrs, but I've been able to make many overlap extensions work perfectly with this method that did not work using traditional pcr.
If possible, use nested primers for the flanking primers in the second round of overlap extension. It helps, but it may not be essential.