After testing with different mix and trouble shooting, I have amplify the ~6kb gene (cDNA) from plasmid and clone into pGEM-T. After sequencing with T7 and SP6, I found the gene specific primer but between these primer the sequence is not desired gene.
It is noted that when I amplified the gene from plasmid there was also a non-specific band that was more stranger than the desired one.
Can anyone provide me your valuable suggestion what is going on with my cloning!!!!
Hi Masuder,
I am assuming that you have not purified the band that you want from an agarose gel, but have just run a PCR/DNA clean up to remove the primers, etc.
From the information you have provided it would appear that you have cloned the stronger band that you see. This is not surprising as a stronger fragment will have more DNA ends which will mean that the stronger has a higher chance for insertion than the one you want.
You don't say how many colonies you have screened, if you have many unscreened it is possible that you have the band you want in there somewhere. Screening them all might find what you want.
The stronger band is from a miss-priming event, as you know some of the sequence now, you can check to see where it if from. To get around this you can purify the band you require from an agarose gel before cloning into pGEM-T. If there is very little insert you could use this product as the template for a second PCR (assuming that the miss-priming is not within this fragment).
-Richard
Hi,
if I understand you correctly then it was your aim to amplify a 6kb cDNA fragment from a plasmid to put it into pGEM-T. After PCR you observed a (smaller?) unspecific PCR product but extracted the correct 6kb product form the gel and cloned it. The obtained plasmid did not contain the desired fragment.
Most likely you cloned the (smaller) side product which will always be present (in a small amount) in the (larger) extracted DNA-fragment but will be cloned more efficiently. You have several options: (i) design new primers, (ii) analyze the sequence of the unspecific product to find a restriction site that is specific for this DNA and pre-digest your PCR product to eliminate the side product or (iii) look for restriction enzymes to cut out the 6kb fragment from the original plasmid and clone it into digested pGEM-T.
Good luck
Hi Masuder,
I've been thinking a bit more about you problem.
Another possibility is that you are only getting miss-priming. You hint that it is a problematic PCR and that you have done some optimisation of the conditions. One thing that might be going on is that you have in fact de-optimised the condition leading to miss-priming and generation of a false positive.
To test that your fragment is what you want, you can sequence the PCR product directly to see what you have got.
Another option for you PCR is to split it into parts, say 3x 2000bp fragments, that you can then assemble together. This will help identify if you are having any problems with the primers.
-Richard