|Register||Search||Today's Posts||Mark Forums Read|
|Molecular Cloning Forum 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.|
| ||LinkBack||Thread Tools||Display Modes|
About the sequencing using topo cloning vector
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?
I have to admit that i don't know much about genetic engineering stuff. so any of your suggestions would help. thanks.
|cloning , sequencing , topo , vector|
|Thread||Thread Starter||Forum||Replies||Last Post|
|Directional TOPO Cloning||kdawg||Molecular Cloning Forum||6||04-04-2013 11:42 PM|
|problems with topo ta cloning||ihatetopo||Molecular Cloning Forum||2||10-19-2010 11:40 PM|
|agarose gel purification of cloning email@example.com||Protocols and Methods Forum||5||06-17-2010 09:33 AM|
|TOPO cloning problems!||annabcn||Molecular Cloning Forum||1||09-22-2009 07:04 AM|
|DNA Cloning Protocol||moleculardude||Molecular Biology Articles and Protocols||2||08-05-2007 02:31 PM|