I am currently cloning a gene of interest (460bp). I amplified my gene of interest using two polymerase 1) NEB Q5 polymerase enzyme 2) Taq polymerase. The forward primer has XHOI restriction site, and the reverse primer has NOTI restriction site. After amplification of my insert, I PCR purified the product and proceeded further to double digestion. For double digestion I used XHO1 and NOT1. After double digestion the product generated from Taq polymerase yielded digested product whereas double digestion of product yielded from Q5 polymerase seems like uncut. What will be the possible problem here. How to trouble shoot this?
Note: I have performed both sequential digestion and double digestion for Q5 polymerase generated amplicons.
Thanks, in advance.
I don't think you can make that conclusion from this gel. I presume the restriction cleavage sites are at the ends of the fragment, so you are looking at a very small mobility different. In fact on the left side the undigested looks the same as the Taq fragment (whereas if you were correct it would look more like the Q5). I think the small differences you are seeing are probably due to differences from different loading amounts.
did you include 3 extra bases at the 5'ends of the primers after the cut sites. Many enzymes need extra random bases for enzyme binding before the cutting takes place. It is possible that the enzyme(s) have not cut
Hi Dr. Rutland, I have added extra bases. When I used the TAQ polymerase amplified product the digestion was successful but, when I used the Q5 polymerase amplified product my digestion product was larger than the undigested product.
Hi Dr. Benedik, Thank you for your insight. I proceeded to ligation and transformation using this product but the ligation was unsuccessful.
If the larger size is a real effect then the only suggestion that I have is that some protein in the q5 mix is still lightly bound to the amplimer . You could try adding a very small amount of SDS to the amplimer before running on the gel to see if the q5product runs any smaller but on the whole I agree with Michael J. Benedik
Thank you Paul Rutland. I will try according to your suggestion.
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