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.

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