Hi all,
I have been working on amplifying two different genes within the same reaction. Earlier while standardizing the conditions and reaction composition I have utilized primers which aren't adapter linked. These adapters are from Illumina.
After standardizing I have ordered a set of primers which are linked with adapters. Since then Duplex PCR is not giving satisfactory results as only one gene is getting amplified while the other is not.
For context I have tried single plex reactions for both the genes with the same standardized conditions and both genes are getting amplified in single plex reactions. The primers I utilized here are adapter linked primer.
I have tried everything I can which include increasing the primer concentration, changing the primer ratio, modifying the annealing temperature, DNA template concentration, dNTP concentration. Nothing worked out in the end.
In summary adapter linked primers are not working for a gene in Duplex PCR. What could be the reason for this and any suggestions to overcome this are welcome :)
Perhaps one primer from each pcr is forming a primer dimer.Then the real pcrs become less efficient ( maybe the failing one is higher gc,melts badly so amplifies badly) so you only see one product. Do you have a fuzzy band at less than 100bp in the duplex pcr?
Can you amplify them singly and then mix the pcr products if mixing is needed.It sounds like they both work well on their own.
Hi Paul Rutland. As you said mixing is within our possible solutions but it is our last resort. There is no fuzzy band at less than 100bp in the duplex PCR. Initially we thought that one of the reagents is consumed very quickly but that is not the case at all as we increased every consummable within the limits and still in Duplex conditions only one product gets amplified.
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