User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/19

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Preparing Parts for LCR

Purpose: 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

  • 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 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)

  • 15uL MV10
  • 2uL 10X T4 PNK Buffer
  • 2uL 10mM ATP
  • 1uL T4 PNK

20 uL rxn for ATF2:

  • 2uL ATF2
  • 2uL 10X T4 PNK Buffer
  • 2uL 10mM ATP
  • 1uL T4 PNK
  • 13uL Water

20uL rxn for Gal4DB_mCh:

  • 2uL Gal4DB_mCh
  • 2uL 10X T4 PNK Buffer
  • 2uL 10mM ATP
  • 1uL T4 PNK
  • 13uL Water

Incubate all three reactions at 37C for 30min. Clean up with PCR clean up kit

Final concentrations in ng/uL:

  • MV10:25
  • ATF2:16
  • Gal4DB_mCh:13
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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


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