sequencing trouble

[doorsea]

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

[CharonY]

It will depend a bit on the nature of your unspecific products (e.g. are they bigger than expected, or smaller?) and what their source is. Is the colony clonal, for instance (i.e. are the different plasmids in your isolation).

As long as the regions are not highly repetitive high AT should not be a problem.