User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/03

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MV10 digest and blunting

Enzymes researched to create new protocol, important notes: Mung Bean Nuclease is active at 30C, will begin to degrade DNA at higher temperatures, must dephosphorylate vector and phosphorylate inserts for LCR, cutsmart best compatible buffer for both enzymes

Purpose: to open up MV10 vector and blunt the ends for subsequent Ligase Cycling Reaction

Methods:

Digest MV10 (concentration: 266ng/uL) with XbaI

MV10 10uL XbaI 1uL Cutsmart Buffer 2uL Water 7uL

Incubate at 37C for 15min (fast acting enzyme) Heat inactivate at 65C for 5min Cool to RT for 10min Add 2.6ul MBN (1u of enzyme for each ug of template DNA) once tube is cooled Incubate at 30C for 30min Clean up with Qiagen PCR clean up kit, elute in 30uL of Elution Solution

Results:

Concentration of vector: 81ng/uL

Image:9.4.15.MV10dig.jpg

This also absolutely huge image shows clearly different banding patterns for the cut and uncut plasmids. We will proceed with LCR.

Note from 9/29/15: Top bands on both are relaxed circular DNA. Bottom bands are supercoiled plasmid. Middle band on cut get should be desired cut product as it is the linear band. Also Rene says to stop using so much dang ladder, that's why its smearing. Thanks Rene.



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