I cloned 900 bp insert in Topo Pc DNA 3.1. After transformation I got few positive clones. I checked these clones by colony pcr and I got the expected size by using forward and reverse primer of vector.
I did plasmid prep and again checked with pcr reaction I got the expected size. I did sequencing with both forward and reverse primers of vector. The sequence from forward primer of vector was fine but sequence from reverse primer was not readable . I tried twice and changed the condition. Further I did sequencing with this reverse primer with a control plasmid and it worked. Can anyone suggest whether this is due to a problem in my clone? Or any possible suggestions.
Is the forward read long enough to check whether the reverse priming site is intact?
When you say it's not too long you mean it's not long enough, or that it is unusually short? If the latter, your sample might simply not be clean enough for Sanger sequencing. These days getting reads 1 kb long is the norm -at least in my experience.
Can't you ask the staff at the sequencing facility to do a primer walk for you? Failure to amplify with the reverse primer is certainly cause for concern.
What reverse primer are you using, BGH?
Primers are cheap. Design a new forward internal primer that is specific to you ORF. Do it ~150 bp before where your current forward primer stops.
Thanks Martin and Burke i repeated the cloning experiment and got the results !
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