I am working on Gateway cloning and using primers that contain attB sites. The primers are 49–50 nucleotides long, and their melting temperatures (Tm) are approximately between 70–76 °C. However, when I perform PCR using Q5 High-Fidelity DNA Polymerase, I am unable to obtain any bands. Interestingly, the same primers without the attB extensions work well with Taq polymerase.
Is there any specific condition or protocol adjustment required for efficient amplification of attB-containing primers with Q5? I would really appreciate any suggestions or tips. Thank you!
Büşra Yirmibeş ,
Some troubleshooting point:
The attB primer is significantly longer (49-50 nt) and has a higher Tm (70-76°C). Therefore, Q5 polymerase has stricter annealing conditions. The attB sequence that does not participate in binding in the long primer will "drag down" the effective annealing region, resulting in the overall calculated Tm not matching the effective binding Tm.
The attB extension part does not form complementarity with the template, which may affect the annealing and primer binding efficiency, so the high-fidelity enzyme does not work. The Taq polymerase has a more tolerant thermal stability and annealing conditions, making it easier to obtain products.
It is recommended that you adjust the PCR program:
1. Initial denaturation: 98°C, 2 minutes
2. Cycle 30 times:
Denaturation: 98°C, 10 seconds
Annealing/extension: 72°C, 30 seconds/kb
3. Final extension: 72°C, 2 minutes
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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