User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/04

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LCR

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:

Begin by phosphorylating the parts. I will try two reactions, one with phosphorylated bb and one with unphosphorylated bb. Couldn't find buffer compatibility info between PNK and Ampligase but buffers appear very similar, won't clean up between phoshphorylation and LCR this time.

90fmol of each part was phosphorylated as follows:

Phosphorylation RXN 1 (set up two of these)

  • 4uL MV10 (81 ng/uL)
  • 1.5uL Gal4DB_mCh (46ng/uL)
  • 1uL ATF2 (56ng/uL)
  • 1uL 10X T4 PNK Buffer
  • 1uL 10mM ATP
  • 1uL T4 PNK
  • .5uL Water


Phosphorylation RXN 2 (set up one of these)

  • 1.5uL Gal4DB_mCh (46ng/uL)
  • 1uL ATF2 (56ng/uL)
  • 1uL 10X T4 PNK Buffer
  • 1uL 10mM ATP
  • 1uL T4 PNK
  • 3.5uL Water

Incubate all three reactions at 37C for 30min. Inactivate at 65 for 20min


LCR

Set up three LCR reactions (30uL each)- one with phosphorylated MV10, one with unphosphorylated MV10, and one no bridges

LCR1:

  • Ampligase 1uL
  • Ampligase Buffer (10X) 3uL
  • DMSO (8%v/v) 2.5uL
  • Betaine (.45M final) 2.5uL
  • bridge 1 (30nM) 3uL
  • bridge 2 (30nM) 3uL
  • bridge 3 (30nM) 3uL
  • Phosphorylation RXN1 10uL
  • Water 2uL

LCR2:

  • Ampligase 1uL
  • Ampligase Buffer (10X) 3uL
  • DMSO (8%v/v) 2.5uL
  • Betaine (.45M final) 2.5uL
  • bridge 1 (30nM) 3uL
  • bridge 2 (30nM) 3uL
  • bridge 3 (30nM) 3uL
  • Phosphorylation RXN2 10uL
  • MV10(81ng/uL) 4uL

LCR3:

  • Ampligase 1uL
  • Ampligase Buffer (10X) 3uL
  • DMSO (8%v/v) 2.5uL
  • Betaine (.45M) 2.5uL
  • Phosphorylation RXN1 10uL
  • Water 11uL

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, H20 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 1.5hrs
  • Plate on LB+Amp (Spin down, remove 200uL of supernatant, resuspend and plate 100uL)
  • Incubate overnight at 37C


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