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Hi all,
I have been working on two genes which I want to sequence later on. Initially I tried amplifying them using Duplex PCR protocol. The primers I utilised initially were not linked with illumina adapter linked. At that time both were amplifying properly. Just for clarification the primers I utilise are universal primers
Later on, I ordered a new set of primers which are linked with illumina adapters. Since then, every time I set up a new reaction only one of them gets amplified, there in no sign of other gene getting amplified. I tried changing the primer ratio, increasing the primer concentration, changing the annealing temperature by +-5C, changing the dNTP concentration, increasing the dNTP concentration. Nothing worked.
Is there any possible reason on why this might happen and any suggestions on how to overcome this are welcome😁
I have been working on two genes which I want to sequence later on. Initially I tried amplifying them using Duplex PCR protocol. The primers I utilised initially were not linked with illumina adapter linked. At that time both were amplifying properly. Just for clarification the primers I utilise are universal primers
Later on, I ordered a new set of primers which are linked with illumina adapters. Since then, every time I set up a new reaction only one of them gets amplified, there in no sign of other gene getting amplified. I tried changing the primer ratio, increasing the primer concentration, changing the annealing temperature by +-5C, changing the dNTP concentration, increasing the dNTP concentration. Nothing worked.
Is there any possible reason on why this might happen and any suggestions on how to overcome this are welcome😁
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