User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/25

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

LCR/PCR Test

Purpose: Re-running LCR to creat MV10_ATF2_Gal4db_mCh construct, testing idea of coupled reaction

Methods:

Create 10X mastermix as follows for 1 RXN of 13uL:

  • Ampligase 1uL
  • Ampligase Buffer 2uL
  • Bridge 1 (300nM) 1uL
  • Bridge 2 (300nM) 1uL
  • Bridge 3 (300nM) 1uL
  • MV10(XbaI,MBN, PNK)(25ng/uL) 2uL
  • ATF2(16ng/uL) 2uL
  • Gal4DB_mCh (13ng/uL) 2uL
  • Water 1uL
  • Bridge1:LCRb_MV10tctGal4DB_rc
  • Bridge2:LCRb_mCh_ATF2_rc
  • Bridge3:LCRb_ATF2tcaMV10_rc

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

Set up the following reactions:

  • Reaction1: normal LCR, add 0.5uL 100%DMSO, run LCR only/transform
  • Reaction2: LCR+PCR, add 3uL GoTaq, run with LCR only
  • Reaction3: LCR+PCR, add 3uL GoTaq, run with LCR then PCR
  • Reaction4: LCR+PCR, run LCR, then add 3uL GoTaq and run PCR
  • Reaction5: LCR+PCR, run LCR, clean up, add GoTaq and run PCR
  • Reaction6: Same as reaction 2, but add 1uL primer P0002
  • Reaction7: Same as reaction 3, but add 1uL primer P0002
  • Reaction8: Same as reaction 4, but add 1uL primer P0002
  • Reaction9: Same as reaction 5, but add 1uL primer P0002
  • Reaction10: Same as reaction 1 but w/ XbaI/MBN unphos MV10 (35ng/uL), run LCR only/transform
  • Reaction11: Same as reaction 1 but w/o bridges + 3uL extra water, run LCR only/transform

All PCRs run with standard GoTaq protocol, 3.5min elongation time, 65C annealing temp

All parts in Reactions 1-9 are phosphorylated and cleaned (is this step unnecessary??)

P0002=12.5pmol/uL

Samples 5 and 9 cleaned up with Qiagen PCR clean up kit and eluted in 10uL elution solution

All stored at 4C overnight


Personal tools