For luciferase assay I want to clone my gene using Kpn1 and Nhe1 as both of these are present in vector but not insert.My clone of interest is human genomic DNA.I have designed my PCR primers using those two restriction sites for amplyfing my region of interest.Form NEB, I got the information that Nhe1 is affected by some combinations of methylation overlapping.But the problem is I have to choose both of these enzymes for cloning and now I am little bit worried about the methylation problem.How can I avoid this problem during digestion?
Hi,
Like it is mentioned, you are PCR amplifying your DNA which eventually will not have methylation. We use these enzyme combinations in the lab. Just follow the appropriate enzyme buffers and units of enzyme as per guide and you are good to go. Sometimes, the strain of bacteria you use for cloning may restrict methylation related issues of single digestion or no transformants. If you don't get transformants, you can use a different strain of bacteria. Check NEB guides of trouble shooting if you get stuck or follow Maniatis protocols.
I just realized its for reporter, I suppose you may be using pGL3 vector for cloning. Refer to the reporter protocol guide for most suitable host for your transformation.
The chart of NEB on methylation (https://www.neb.com/tools-and-resources/selection-charts/dam-dcm-and-cpg-methylation) includes NheI ( Nhe1 is incorrect, then number of a RE is usually a Roman numeral). You can find there NheI is sensitive to methylation of some CpG overlaps. It is insensitive to Dam or Dcm. The latter are phenomena that happen in prokaryotes (i.e. bacteria) while CpG methylation occurs in eukaryotes.
Did you clone your vector from bacteria, you won’t run into trouble cutting with NheI.
Why don’t you just give it a try? Include single digestions (only KpnI and NheI) and a combination of both on a small sample. Don’t forget to include a sample with no RE, but incubated in the buffer! Which buffer are you planning to use for the double digestion?
Good luck
Duco
Hi Duco,
I will clone my vector in bacteria and use NEB Buffer1:1
PCR amplification of DNA should not have methylation. I've used these enzymes (High Fidelity version) with CutSmart buffer and they work pretty well.
Good luck !!!
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