Barrick Lab :: ProtocolsDpnIDigestion

---+++ Dpn I digestion

*Purpose*

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

*Use*

1. Add 1µL of [[https://www.neb.com/products/r0176-dpni][DpnI]] to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix. <br /> 2. Incubate the mixture at 37°C 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). <br /> 3. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!

<strong>Other Notes</strong> 

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 80°C for 20 minutes.

*Procurement*

Can be ordered directly from NEB. <br /> Typically stored at -20°C; 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 µg of pBR322 DNA (dam methylated) in 1 hour at 37°C in a total reaction volume of 50 µl.

NEB buffer composition:

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

Barrick Lab > ProtocolList > ProtocolUpdatesNeeded > ProtocolsDpnIDigestion

Contributors to this topic

SeanLeonard, CraigBarnhart

Topic revision: r4 - 2017-11-06 - 18:03:43 - Main.SeanLeonard