User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/03: Difference between revisions
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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 | |||
Revision as of 12:26, 4 September 2015
 Project name
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MV10 digest and bluntingEnzymes 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
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