Do I have to perform Dpn1 digestion (of the template strand) immediately after the PCR reaction or can I store the PCR product at -20 and then do the Dpn1 digestion after maybe say 2 or 3 days?
Dear Sujeet,
assuming you want to destroy the methylated PCR template, the DpnI digest should work independently of storage. Beware however that the polymerase will happily fill in nucelotide leftovers from the PCR and create blunt or A overhangs which might interfere with subsequent cloning. A simple precipitation with alcohol or a PCR cleanup spin column procedure before the DpnI digest will overcome these potential problems.
Dear Sujeet,
Please note that Dpn I can cleave dsDNA templates only when its recognition site is methylated. Since PCR amplicons lack methylation, they are not suitable templates for Dpn I. You may check this with your PCR amplicons in a pilot scale. I suggest you to find another enzyme for your amplicons, which do not need methylation or any other DNA modification for cleaving.
Rest you can easily store your PCR amplicons at -20C for RE digestion at a later stage. We had good results even after cutting the amplicons after 1 year.
best wishes,
SD
Dear Sujeet,
assuming you want to destroy the methylated PCR template, the DpnI digest should work independently of storage. Beware however that the polymerase will happily fill in nucelotide leftovers from the PCR and create blunt or A overhangs which might interfere with subsequent cloning. A simple precipitation with alcohol or a PCR cleanup spin column procedure before the DpnI digest will overcome these potential problems.
It will be no problem to store you PCR rxn before digestion.
You can add the DpnI enzyme directly to the PCR reaction after cycling and cooling to below 37ºC. It works fine in the PCR buffer. However, as noted by Wolfgang the pol will fill in the ends.
- Badges
- Science method