(r2) Barrick Lab :: ProtocolsDpnIDigestion

---+++ Dpn I digestion

*Purpose* <br>

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

*Use* <br>

1.  Add 1µL of [[https://www.neb.com/products/r0176-dpni][DpnI]] to 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!  <br>


*Other Notes* <br>

Use in NEBuffer 4; however, it can be added directly to PCR sample.<br>

Heat inactivate by incubating at 80°C for 20 minutes.<br>

*Procurement* <br>

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

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 <br>

Barrick Lab > ProtocolList > ProtocolUpdatesNeeded > ProtocolsDpnIDigestion

Contributors to this topic

SeanLeonard, CraigBarnhart

Topic revision: r2 - 2014-07-21 - 21:31:48 - Main.SeanLeonard