1. I always get no signal at partial region of promoter of pCAGEN after sequencing. I have tried couples of primer before (forward))/after (reverse) that region but it still can't work.
Portions of the pCAGEN promoter are extremely GC rich, which can complicate primer design and which may interfere with replication. You may wish to check the GC% of the region your amplifying and your primer annealing sites. I have a list of pCAGEN genotyping primers I got to work for one mouse project, but it would take some time to dig it up.
Dear Pin-Chun,
If you can set a regular end-point PCR with Fw/Rv primers you mention, can you amplify all the extension of the promoter (meaning getting a band equal to its known size)? It could happen that during the sequence reaction, the polymerase encounters regions with strong secondary structure within the promoter causing the polymerase to "fell off". It may also happens that the sequencing reaction yields sequenced products that to short (reliable up to 1000 bp in average) compared to the length of the CAG promoter in the pCAGEN (aprox 1700 bp).
I would try to design a couple of internal Fw/Rv primers for the promoter to help cover the whole extension of the promoter
Best,
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