I used a proof reading DNA polymerase, Q5, to amplify backbones and inserts that must have correct nucleotide sequences. I have already excised the bands at the appropriate lengths and have purified my products. I would like to rid the DNA of the template DNA as it is a contaminant that I would like to avoid growing. Can I simply use a dpn1 digest to remove this template DNA and subsequently perform Gibson Assembly? Is this also possible after Gibson Assembly?
Thank you!
Dear Draven
if your template is a methylated dna as for example a plasmid propagated into bacteria you can certainly use dpni to digest it.
Also if you already perform dna extraction from agarose gel and your template size is different (e.g. higher) then the per product size probably you already remove the template, however dpni digestion is simple, fast and could be performed by adding enzine and buffer directly on pcr sample.
I routinelly using it to remove background due to template in PIPE cloning. if you are interested to know more about it, you can found a presentation on my blog ProteoCool https://proteocool.blogspot.com
good luck
ciao
Manuele
I have routinely done a 50 µl Q5 PCR and after it finished, just opened up the tube to put in 1 µl DpnI, and then ran the digest on the thermocycler after mixing.
Draven Rane
Manuele Martinelli is right that if your template was larger than your PCR you don't have to use any DpnI because you did gel purification. Otherwise, you should start your PCR with 2-5ng of the template, run 5ul of your PCXR reaction on the gel to see that your PCR worked. Take other 45ul of your PCR, add 1ul of DpnI and put on 37C for couple hours. DpnI is not very good enzymes, it looses activity very fast. So use fresh DpnI and use 2-5ng of the template, as I already said
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