Checking PCR insert (into pGEMT vector) - (Jan/07/2010 )

Hi,

I have been having trouble screening whether I have my small PCR product (300bp) inserted into pGEMT vector.
I generated the PCR using primers with XbaI and ApaI sites, then I ran the PCR products on a gel, extracted the products, did A-tailing, gel purified again, then cloned the fragment into the pGEMT vector followed by transformation into DH5alpha.

I picked the white colonies (And also did a negative control by transforming empty pGEMT vector to DH5a, in which I got only blue colonies) -- did miniprep and ran a digestion using XbaI and ApaI.... and I didn't see the expeted 300 bp fragment !!! However, when I PCR these minipreps using the primers (that I used to amplify the fragment), I got the band with the right size. Which suggests the restriction sites didn't get incorporated into the vector?? So now I'm quite confused with the result!!! If anyone can help that'd be great !!!! Thanks!!

annlin on Jan 8 2010, 01:25 PM said:

You should provide more detail, so the problem can be seen more easily. I have some comments based on what you mentioned:
1. Can you confirm the sequence of your primer (the mistake of the primer producer or maybe your design), so check that from your experiment design one more time.
2. Did you incubate separately the plasmids in 25 degree with ApaI and then 37 degree with XbaI? (ApaI is active in 25 degree) Did you use the suitable buffer for both of them (it should be buffer 4 of NewEngland Biolab)?
3. Did you check the enzyme activity? (digest one of your plasmid with ApaI by excess enzyme amount and see whether only one band appears on the gel, because there is an ApaI on pGEM-T vector; digest a plasmid has XbaI site as positive sample).
I strongly suspect that your digestion condition was not right or your ApaI was not that active (because in TA cloning, there are chances that you have correct clones with reversed orientation of your PCR product. So, if the ApaI is active, you should see some of your samples with expected band because of an ApaI located near the incorporation site on pGEM-T)

You could also have accidentally created an XbaI site which is methylated in standard cloning strains. You must avoid the sequences TCTAGAtc or gaTCTAGA because the methylation of the GATC site will inhibit XbaI digestion. There may be similar issues with your other enzyme.