We prepared a PCR buffer and assessed it with approximately 12 primer pairs, all of which resulted in successful amplification. However, there was a specific gene region that failed to amplify. The primers work well and efficient with Ampliqon 2x red PCR buffer resulting in the aplification of our desired targets.
The picture shows the PCR products attained from both Ampliqon and our custom PCR buffer for two DNA samples (performed in duplicate).
Various additives including DMSO and PCR enhancers were used, yet none proved effective.
I welcome any suggestions that could assist in resolving this matter.
Ampliqon red buffer is a high ammonium containing buffer. What is the recipe for your homemade buffer. Also the amount of magnesium can make a difference as the annealing temperature of primers is sensitive to divalent ion concentrations. You should also check for snps at the 3' end of each primer since most of your primer pairs do work so primer sequence could be a possibility
When I look at the products in the third, fifth, seventh, and ninth wells from the left (including the ladder), I see at least three products of different sizes. This suggests that the primers may be reacting with each other (which is quite common). Then, there is a product of about 100 bp, which aligns with the first band of the ladder (assuming the ladder is 100 bp), and then products of higher base pairs. The difference between the third, fifth, seventh, and ninth wells may be due to varying concentrations. Try loading more of your PCR products for the samples pipped into the third and fifth wells. If the difference is still not seen, consider adding more DNA to your PCR reaction. What is the concentration of your DNA samples (ng/µL)? Additionally, please keep in mind the previous suggestions by Mr. Paul Rutland.
Thanks Paul Rutland to share your opinion with me. Our home made buffer is KCL base. I thought that this might be effective to test NH4 base buffer. I will share the result with you.
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