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
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