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Sequencing with universal primers

Sequencing with universal primers - Molecular Cloning Forum

Sequencing with universal primers - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 10-26-2011, 05:32 AM
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Cool Sequencing with universal primers



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
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