I have been trying to linearize my backbone vector by PCR followed by digestion with Dpn1 restriction enzyme to remove methylation sites before ligation.
After PCR, I did gel electrophoresis and I could see the bands meaning my PCR was successful but after digesting with Dpn1 I could not see any bands.
My reaction set up was as follows: 50ul PCR product +2ul Dpn1. Incubated at 37 degrees for 1 hour.
In what situations must I digest the plasmid vectors with Dpn1?
Hello Caroline,
It sounds odd. Dpn1 cuts when its recognition site is methylated (it cuts so it doesn't remove methylation sites). Therefore, it will cut the original template. I would try to get new reagents just in case and try again (fresh sterile water, buffer, and if it doesn't work, change enzyme)
Where is this plasmid coming from? If it is coming from bacteria, is it dam+?
I hope it helps.
All the best,
Roberto
Hello Roberto,
Thank you for your feedback. I am quite new at this so I am definitely sure my question may not be framed well. Nonetheless, I definitely need to confirm the plasmid origin.
Your experiment is slightly confusing. As Roberto de la Cerda mentions, DpnI is a restriction enzyme that cuts DNA at methylated GATC sites. It is frequently used to remove methylated template DNA and leave behind unmethylated templated. So if you are amplifying your plasmid (and linearizing it) then treating with DpnI, the original template should be removed by the DpnI but any PCR product should not be digested.
Are you sure that you saw the full length linearized plasmid backbone you were looking for when you ran your gel? I am confused when you mention bands because I would think there should only be a single band.
Hi.
I would like to add my personal comment here. I use the same strategy for Site-Directed Mutagenesis to cut off the methylated template DNA (PCR followed by DPN1 digestion 30 ul DNA, 2.5ul Dpn1, and 1.5-3h digestion at 37 degrees C. Further, I do not load the digested plasmid again on the agarose gel. I transform it further into DH5alpha cells and obtained positive clones (confirmed by Sanger sequencing). While I am not sure why doesn't it show up (have not seen it), you can give it a try by transforming the digested product and see if you get colonies the next day.
Hope it helps.
Sonal Jaiswal Thank you so much for your response. I will also try to do that and see if I get positive clones.
You should add in the restriction digest buffer to a final concentration of 1X. I know it's not strictly necessary, but it does help.
If the enzyme is heat-inactivated, add in a hold at high temp step. See the product insert for suggested time and temp.
Good luck!
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