I am having problems amplifying my insert; basically, following the completion of the PCR reaction I can only visualize intense bands on an agarose gel for 2 of the 4 constructs (These 4 constructs only differ by 1 nucleotide).
Below I will describe the reaction I'm using and some of the ways I've tried to troubleshoot. I would GREATLY appreciate any additional suggestions or comments you may have: Reaction
3ul 10x Pfu Buffer
1ul dNTP mix
1ul construct (50ng/ul)
1ul forward primer (30uM)
1ul reverse primer (30uM)
1ul Pfu Turbo
30ul TOTAL PCR Program
94 degrees for 2min
94 " for 1 min
51 " for 1 min
72 " for 3 min
72 degrees for 10 min
4 " forever PCR Product Purification
Add 150ul PB (Qiagen)
Load on a Qiagen extraction kit spin column
Spin for 1min at 13000 rpm
Wash with 750ul PE (Qiagen) and 1 min spin
Remove flow-thru and 1 min spin
Add EB and elute DNA
So, all four constructs use the same primers, buffers, polymerase, etc. Troubleshooting myself I have found that the 2 constructs with only light bands work better at 57 degrees (step 3) and at 40 cycles instead of 30. In addition, I have also transformed some of my starting construct. These adjustments, however, do not help because following PCR purification I still have a digestion and gel extraction to complete. Following digestion and running the DNA on a 0.8% agarose gel, I cannot visualize any bands. The intensity of the bands (DNA concentration) for the other 2 constructs, however, was high enough to successfully complete a ligation.
What I'm trying to understand is: what is different about these two groups of plasmids and what can I do to fix my amplification dilemma?
Thank you for your help!