Restriction digestion of clones give undesired product - (Sep/19/2011 )

i have cloned a 2.6 Kb insert into pGEM-T easy vector . prior to that i had done a tailing of the PCR product and gel purified the band . hen proceeded with TA cloning and after transforming 4-6 colonies .among them i screened 4 and isolated plasmid from all . after plasmid isolation i cut digested it with the particular restriction enzymes namely - Kpn1 and Bsu361 . incubated it 3 hours at 37 C . i heat inactivated the product after the required time . and run them on 0.5 % gel-
ideally i should have got inserts of 2.6 kb in all of them . but i got it only in one of them . and in the other i got bands at around 1.5 KB .

my question is what is wrong ?


how can T vectors give transformed colonies if they r not ligated ?
if they have ligated it should b the product i have gel purified then Wat is the band around 1.5 ?
has there been a wrong insert ? but how is it possible i did gel purification and reconfirmed the band by running it on gel and getting desired band .

Is there a restriction site for one of your enzymes in the PCR product? Does one of the RE's exhibit star activity?

What controls did you run for your ligations?

there is no restriction site in the PCR product
none of Kpn 1 and Bsu 361 are reported of star activity- i checked that ..
i did not run any control for ligation .. why would that be necessary?

As far as I can tell, pGEM-t doesn't have either Kpn1 or Bsu361 sites - unless you added these sites, there shouldn't be any cutting of the plasmid.

The vector can self ligate - this is quite common with complementary ends.

Perhaps your PCR product wasn't what you thought it was. Try sequencing the vectors you have and see if there is a problem or not.