PCR problem from transformed TOPO TA vector - (Jul/02/2009 )
Hi
Presently I am trying TOPO TA cloning of 280bp insert. I've got white colonies and I've isolated plasmid as well. Then I've performed PCR using M13 forward and reverse primers. But I ended up with smears and multiple bands. Then I've tried my insert specific forward primer (which is degenerate) with M13 reverse. I've obtained single band, then I purified the band from gel and performed PCR using my insert specific forward and reverse primers (Both are degenerate). I've found no band in agarose gel.
But I've got single band in combination of my insert specific primer with M13 reverse from the gel purified product. I've tested the PCR reagents and primers. They are Ok.
Please help me....
Have you tried sequencing the TOPO clones with M13F and M13R to see if they have your inserts? If you miniprep them and do sequencing, you can find the clone you want and work from there.
cloneguru on Jul 2 2009, 12:52 AM said:
Presently I am trying TOPO TA cloning of 280bp insert. I've got white colonies and I've isolated plasmid as well. Then I've performed PCR using M13 forward and reverse primers. But I ended up with smears and multiple bands. Then I've tried my insert specific forward primer (which is degenerate) with M13 reverse. I've obtained single band, then I purified the band from gel and performed PCR using my insert specific forward and reverse primers (Both are degenerate). I've found no band in agarose gel.
But I've got single band in combination of my insert specific primer with M13 reverse from the gel purified product. I've tested the PCR reagents and primers. They are Ok.
Please help me....
Same as above. Perform sequencing. Also sequence the ISP/M13 unique product.
Also, you can digest the clone/s to see insert size. Not as accurate, but will give you some encouragement if you see one.
Cellcounter is right, the digest will give you some form of encouragements. I have had cases whereby the inserts on the gel were all wrong. Once, I had inserts of the right size, but they turned out to be artifacts. To save time, do your digest and sequencing at the same time.
Hope everything goes well for you. 