Dpn I digestion

Purpose

To digest (Adeno) methylated GATC sites. Useful for removing cell-derived plasmid template from PCR samples.

Use

1. Add 1L of DpnI to finished 50L PCR reactions (or .5L to 25L reactions). Pipet or invert to mix.
2. Incubate the mixture at 37C for 1-2 hrs (depending on your paranoia or need to remove template DNA). Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
3. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!

Other Notes

DpnI can (and should) be added directly to PCR sample.

Outside of PCR reactions, use DpnI with NEBuffer 4 or Custmart.

Heat inactivate by incubating at 80C for 20 minutes.

Procurement

Can be ordered directly from NEB.
Typically stored at -20C; can be found in the common enzyme freezer box.

NEB unit definition:

One unit is defined as the amount of enzyme required to digest 1 g of pBR322 DNA (dam methylated) in 1 hour at 37C in a total reaction volume of 50 l.

NEB buffer composition:

1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25C

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Topic revision: r4 - 06 Nov 2017 - 18:03:43 - Main.SeanLeonard
 
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