Hello. I am trying to discover the sequences of a primer mix composed by around 80 couple of prmers. My idea was to use a NGS approach to clone the primers using a library but most of DNA libraries require double strand DNA. Any idea how to reach this goal?
Hi stefano
did you try some revercse transcription on your primer pool to obtain a double stranded set?
fred
Try to do a PCR and sequence the Amplicons. Then you know at least, where the primers bind and what they amplify, although you do not know the exact length of them.
the problem is that most of the protocols need a double stranded target in order to ligate adaptators and amplify the library. a RT step is therefore necessary.
fred
What do you not understand? A PCR product is always doublestranded and can be used to ligate the adapters. Do you know what RT is? Reverse transcription. It needs an RNA template and a DNA primers. If you have a PCR primer (which is RNA and not DNA) you need to anneal a DNA primer to start the reverse transcrition... So your idea won't work. Transcriptio DNA template generate RNA, reverse transcription RNA template make DNA.
the best thing is to ligate an adapter and then do a PCR as Andre was suggesting.
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