Sequential digestion - (Mar/01/2007 )

Does anybody know pESC-TRP vector (Stratagene)? I want to insert a 1.4kb fragment from
pGEM-T-vector into pESC-TRP vector. First I did double digestion with Not I & Spe I (in NEB buffer 2) but after running the gel I found just a ‘smear’ appeared (especially for pESC-Trp vector). Even with pGEM-T digested DNA yield was very much reduced (I used the plasmid DNA 5ug for 20 ul reaction). Ofcource, the expected size (1.4kb) band appeared with pGEM-T, but DNA was degraded. Then I did sequential digestion, first with Spe I (NEB buffer 2) then phenol chloroform extraction and ethanol precipitation then with Not I (NEB buffer 3). That also failed. Again I did sequential digestion, first with Not I (buffer 2) O/N and then with Spe I (buffer 2) for 2h. That also failed. What will be the problem? Please anybody help me to sort out this problem?
Actually this two double digest sites in pESC-TRP vector are very close (refer http://www.stratagene.com/manuals/217451.pdf). I didn't use this vector before. But for pGEM-T what will be the problem? Both vectors I successfully double digested with Not I & Bgl II. But for Spe I it did not work. Why? [pESC-trp vector digestion with Spe I image attached].

Thanks.

Binu

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did you run an uncut DNA just to confirm the DNA has not already degraded.

Also try to change buffers for restriction enzymes. Sometimes, they could be contaminated.

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Yes i ran an uncut DNA. It was not degraded. Same uncut DNA I used for Not I & Bgl II digestion & got good result. There is no possibility of contamination of buffer (NEB buffer 2). Anyway let me check & will tell you. Thanks a lot for your suggestion & reply. Binu

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I think ur problem might be with the close sites!!!

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