Can I digest a specific DNA sequence without a known restriction enzyme site in vitro? I am trying to digest a fragment with CGTCAT but not CATCAT. I tried cas9 with a sgRNA containing the target sequence, but It digested the DNA fragment with CATCAT substitution as well.
You can reamplify using ARMS or DCAPS pcr primers if you just need to show the existence of each sequence. I find DCAPS more specific although more work due to the added restriction cutting.
https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/
You can reamplify using ARMS or DCAPS pcr primers if you just need to show the existence of each sequence. I find DCAPS more specific although more work due to the added restriction cutting.
https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/
If you are going to try dcaps Yun Chen then add some (6-8_ non annealing bases at the 5'end of the dcaps primer. It just makes the separation of cut/uncut more obvious if you cut eg 26 rather than 20 bases off the amplimer which should be quite small again to help size identification between cut and uncut.
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