User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/06

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Ligase Cycling reaction for MV10+Gal4DB_mCh+ATF2 and Transformation

Purpose: To build the following construct: https://benchling.com/s/x1UVKVF9/edit. This will be the first of many similar constructs, each with varying activation domains. ATF2 is the trial activation domain in this construct.

Methods:

Set up three LCR reactions (25uL each)- one with phosphorylated MV10, one with unphosphorylated MV10, and one no ligase control

All concentrations listed are final concentrations in the solution. 300nM bridge stocks and 30nM part stocks were created.

Reaction Mix: Ampligase 0.5uL Ampligase Buffer (10X) 2.5uL DMSO (8%v/v) 2uL bridge 1 (30nM) 2.5uL bridge 2 (30nM) 2.5uL bridge 3 (30nM) 2.5uL MV10 (3nM) 2.5uL ATF2 (3nM) 2.5uL Gal4DB_mCh (3nM) 2.5uL Water 5uL

Reaction1: with phosphorylated MV10 Reaction2: with unphosphorylated MV10 Reaction3: with no ligase control

Bridge1:LCRb_MV10tctGal4DB_rc Bridge2:LCRb_mCh_ATF2_rc Bridge3:LCRb_ATF2tcaMV10_rc

Cycle with the following parameters: 2 min at 94C 50 cycles(10s at 94C, 30s at 55C, 60s at 66C) 4C hold


Transformation

5 transformations: LCR1, LCR2, LCR3, H2O only control (-), MV10 plasmid control (+)

  • Add 50uL of DH5alpha turbo cells to each tube with appropriate sample (2.5uL of each)
  • Incubate on ice for 30min
  • Heat shock at 42C for 35sec
  • Incubate on ice for 2min
  • Add 750uL of SOC media (RT) to each tube
  • Shake 37C for 1hr
  • Plate on LB+Amp (Spin down, remove 100uL of supernatant, resuspend and plate)
  • Incubate overnight at 37C


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