User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/19
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Preparing Parts for LCRPurpose: to open up MV10 vector and blunt the ends for subsequent Ligase Cycling Reaction (temperature/time of MBN incubation changed) Methods: Digest MV10 (concentration: 266ng/uL) with XbaI
Incubate at 37C for 15min (fast acting enzyme). Heat inactivate at 65C for 5min. Cool to RT for 5min. Add 2.6ul MBN (1u of enzyme for each ug of template DNA) once tube is cooled. Incubate at RT for 25min. Clean up with Qiagen PCR clean up kit, elute in 50uL of Elution Solution. Final concentration:35 ng/uL Purpose: to phosphorylate parts for LCR Methods: ~90fmol of each part was phosphorylated as follows: 20 uL rxn for MV10:(don't have nearly 90fmol...will use as much as possible in 20uL rxn)
20 uL rxn for ATF2:
20uL rxn for Gal4DB_mCh:
Incubate all three reactions at 37C for 30min. Clean up with PCR clean up kit Final concentrations in ng/uL:
Set up LCR reaction components Make a 300 nM working solution of oligo bridges (final volume = 100 μL) in a new tubes. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL Use 1.0 μL of oligo working sln. per 10 μL LCR reaction Fragments diluted as specified here : http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/06 | |
