If there are nonspecific amplification bands, what can be done to improve specificity?
If there are nonspecific amplification bands, what can be done to improve specificity?
1. All Takara Bio PCR polymerases
-Issue:
Primers are not specific.
-Solution:
Use BLAST alignment to determine if the 3' ends of the primers are complementary to sites other than the target site(s). Redesign primers if necessary or modify PCR conditions.
-Issue:
PCR conditions are not sufficiently stringent.
-Solutions:
*Increase the annealing temperature in increments of 2 degrees.
*Use touchdown PCR.
*Use a two-step PCR protocol.
*Reduce the number of PCR cycles.
-Issue:
Too much template was used.
-Solution:
Reduce the amount by 2–5 fold.
2. PrimeSTAR HS and PrimeSTAR Max DNA polymerases
-Issue:
Annealing time is too long.
-Solution:
To achieve specific amplification, it is essential to use a short annealing time (5–15 sec) when performing three-step PCR.
3. PrimeSTAR GXL DNA polymerases
Issue:
Primers have suboptimal Tm values.
Solution:
To amplify targets <1 kb, design primers with Tm values >55°C, and use an annealing temperature of 60°C. If the primer Tm values are <55°C, try a shorter extension time between 5 and 10 sec/kb.
4. Takara Ex Taq and Takara LA Taq DNA polymerases
-Issue:
Nonspecific primer annealing at low temperatures.
-Solution:
The hot-start versions of these enzymes may improve results for some primers.
5. SpeedSTAR HS DNA Polymerase
-Issue:
Smearing of the PCR product bands on a gel.
-Solution:
Excessively long extension times may result in smearing. The general recommendation for extension time for this enzyme is 10–20 sec/kb. If PCR yield is low, try increasing the number of cycles by 5.