I am using Psy I (is-schizomer of Asp I) and my PCR product didn't get in digested form. Instead I see the only band on the gel is that of my PCR product (i.e 900b.p). I use enzymes from Thermofisher Scientific and I follow the manufacturer's instructions. How do I troubleshoot this issue?
Hello Iqra,
Some suggestions:
- Did you run a control to make sure that your batch of PsyI was still active ? I would try to digest a purified plasmid harbouring a PsyI site with PsyI as well as with another enzyme that is supposed to cut the plasmid.
- Did you make a calculation to make sure that you don't use too much DNA/too little enzyme ? Don't forget that in terms of nmoles/ml, a 900-bp fragment is 5 x more concentrated than a 4.5 kb fragment for the same concentration given in µg/ml
- Are you sure that you amplified the right fragment ? The question may be relevant if you tried to clone some DNA fragment and attempt to identify the insert by PCR using vector-derived primers
- If you know the sequence of your amplicon (most likely you do, since you expect that it contains a PsyI site), you could design another primer derived from that sequence and run a second PCR using your first amplicon as a template to see if you get a fragmenet with the proper size.
- You could column-purify the fragment and send it for sequencing to check if the sequence matches your expectation.
Good luck,
Pierre
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