What will be the difference if one does Dpn1 digestion to the gel excised pcr product and directly to the pcr product? If one does it to the gel excised PCR product, does it will affect the cloning and further steps?
For conventional PCR it makes no sense to cut the PCR product with DpnI. DpnI cuts at the recognition sequence GATC only when the A is methylated. The common E. coli strains methylate this site. When you cut PCR products with DPN1 you destroy the parental strain at all GATC elements. This is used in site directed mutagenesis where you want to get rid of the non-mutated template.
If you are amplifying your gene of interest from a plasmid, sufficient plasmid to get some colonies is available in your PCR mix. To get rid of the plasmid you may use DpnI digestion. You may try gel purifying your PCR product and use it directly for cloning without DpnI digestion (you are separating background plasmid from PCR product by agarose gel electrophoresis). There is a possibility of a supercoiled plasmid moving along with PCR product cannot ruled out. Hence, it is advisable to go for DpnI digestion when your template for PCR amplification is a plasmid.
Agree with the other posts here. If you are performing QuikChange mutagenesis, then you need to use template plasmid that was propagated in a methylation-competent strain of E. coli and digest the template with DpnI at the end of the PCR reaction. Gel isolation is only recommended in QuikChange if you have multiple, differently-size PCR products, and even then may not be essential, as smaller bands often are replication-deficient.
For simplicity, it usually works best to perform the DpnI digest immediately after the PCR reaction. However, technically, it would still work fine after a gel isolation step.
If you are performing PCR-mediated subcloning (not QuikChange), then there is no need to include a DpnI digestion if your PCR product is easily separated from the template DNA based on size.
I also want to know if DpnI can be used in PCR reaction. I hope to cut the original plasmid template so that it does not contaminate downstream cloning, and I think this is also what Debasish Paul wants to do.
I am using Phusion HF PCR reaction. It would be nice if I could just add DpnI directly to the reaction to save several steps.
Thanks,
Nicolce
I found the answer.
https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers
DpnI works well (complete cutting) in Phusion-HF PCR reaction.
I agree with Daniel: it is advisable to do before gel extraction but can be performed after. However, the extraction buffer and remnant agarose can complicate downstream applications, perhaps including Dpn1. Look on a nanodrop or other UV spectrometer at your agarose purified DNA. If there is considerable skewed absorbance (even an extra peak) between 260 and 214, and sometimes greater than 260 nm, then I would use another PCR cleanup kit (you can also use mini prep kits *wink wink*) on the sample before doing anything else with it, including Dpn1 digestion.
The purpose of gel extracted DNA is different rether than the simple amplification of a segment of templates
If you used Taq pol for the final amplification, you may need to change the environment the amplicon is in, as DpnI has been shown to have low activity in it. However, you do not need to add anything else except DpnI in your pcr mix if the reaction involved phusion HF. I have seen that it works equally well in NEB and Thermo Phusion HF buffers.
Usually, after the PCR, I add ~0.4ul of NEB DpnI (w/o cutsmart) to 20ul reaction and leave it at 30-37°C for 3 hours or at RT overnight.
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