Hi guys,
i got some problems with sequencing recently.
i just assembled expression cassette (~3.1kb) with overlap extension PCR, and cloned into a expression vector. Then i used outer primer pair to PCR out the fragment of interest. Although the primers are kinda long (~40bp) because they were designed for OE PCR, I got a quite clean band of interest. So i sent the cloning vector with the primers out for sequencing. but failed. Tell from the trace of sequencing results, whole sequence is non-specific from the start of the sequence. i asked for the help from the company. their support told me maybe my primers were not good. so i cloned the fragment into Zero Blunt TOPO cloning vector (invitrogen) and using colony PCR and enzymatic digestion verified the clones. when i got the positive clones, i isolate the plasmids and used T3,T7, M13 to PCR out the fragment. i did get my band of interst. however, still there are some apparent non-specific bands in PCR products. So any idea what's going on? does this kind of sample work for the sequencing company? By the way, my target fragment is AT-rich (62%). do you think that has negative effect on sequencing?
I have to admit that i don't know much about genetic engineering stuff. so any of your suggestions would help. thanks.
Leo
