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Restriction digestion of clones give undesired product


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#1 Debo

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Posted 19 September 2011 - 02:45 AM

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 .

#2 bob1

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Posted 19 September 2011 - 05:08 PM

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?

#3 Debo

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Posted 21 September 2011 - 02:04 AM

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?

#4 bob1

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Posted 21 September 2011 - 05:02 PM

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.




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