Hello there,
I'm having some issues with digesting a PCR product and using it for ligation with the vector pET-28a.
My PCR product is about ~1200 bp, I purify it and get about 30 ng/uL. Then I digest it using NotI and NcoI enzymes, but when I run the digestion what I get are two bands, one about 1200bp and another about 1000bp (the digestion should result in a ~800 bp product). The digestion using only NcoI or only NotI should get a ~900 bp fragment. I even tried to use that ~1000 bp with the digested pet-28a vector in ligation to see what I would get, but I get no colony (I'm using BL21(DE3)) at all.
Any suggestions?
I am unsure of the logic that says cutting with either one enzyme gives a size of 900 but cutting with both gives 800 but . Not1 is quite fussy about buffer type and may cut at only 10% in the wrong buffer. Have you tried the single digests to show that both enzymes are working and can cut your template. Is it possible that your template has a polymorphism at one of the cut sites disabling good digestion?
It might be an idea to cut the product with a third enzyme only to check that the fragment sizes are correct and that this is the correct amplimer. was the original 1200bp amplimer sequenced?
Hello Rafael,
Have you checked for the basics? Gel concentration, enzymes activity, water quality etc? Sometimes can be simple like that.
Best,
Roberto
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