I have six constructs with the following G-C contents: 29.6%, 23.2%, 27.2%, 24.4%, 27% and 28%. Due to the low G-C content of genes of interest, the Tm of my primers (see attached file) are low; calculated between 45 and 55 °C using the Thermo Scientific Tm Calculator (Taq-based).
I started off with a gradient PCR using the protocol slated below for all six constructs. I had no product but primer dimers appearing as a thick band below the last band of a 1 kb plus ladder. At the end of the day, I had optimized annealing temperatures between 38 °C and 65 °C (First 38-52°C and then 48-62 °C). I played a little with the extension time (increasing) but no luck
Component 50 μl rxn Final Conc.
10X DreamTaq Buf. 5 μl 1X
2 mM dNTPs 5 μl 200 µM
1.25 µM Primers mix 5 μl 0.125 µM
Template DNA 1 μl 2 ng
Taq DNA Polymerase 0.5 µl 2.5 units/50 µl Nuclease-free water 33.5 µl —
Cycling conditions
Step Temp Time Cycles
1. Initial Denat. 95 °C 2 min
2. Denat. 94 °C 30 sec
3. Annealing 38-52°C/48-62 °C (Grad) 30 sec
4. Extension 68 °C 4 min
5. Go to 2 24X
6. Final Ext. 68 °C 10 min
7. HOLD 4 °C —
I increased the Mg2+ conc. and nothing happened. DMSO won't be my friend because of the low GC content I guess :).
I checked the conc. of my ordered genomic DNA to 130 ng/μl on the NanoDrop. I decided to increase the concentration by adding up to 100 ng/50 μl Rxn and nothing. I have read here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145803/ that reducing the extension temperature (to as low as 60 °C) required for PCR amplification of extremely A+T-rich DNA leads to product formation. That did not work. Instead, I had some unspecific product for two of the constructs as well as primer dimers. I have also done a control experiment together to validate the reagents and everything worked very fine with the control. My primer sets (forward and reverse) are mixed in a single-stock aliquot. Could this be the problem?
Please I will greatly appreciate any proposals. Thanks in advance.