Amplifying insert region using ORFeome_Seq_Frd/Rvs.


Protocol

1 Reaction preparation

List of paramater tested.

Tm/Target gene MORF4L1 MRFAP1 BCL2L2 BIK MRFAP1L1
Row/Col on PCR plate 1 3 4 5 6
  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) 1 reaction Volume (µl) for 50 reactions with 20µL vol Final concenration Memo
ddH20 5.12 512 -
Phusion HF buffer (5x) 2.0 200 -
25mM dNTPs 0.08 8 0.2 mM Equal volume mix of 100mM dA/T/G/CTPs will give this solution.
Phusion pol 0.2 20 - Home brew Phusion pol
100µM Frd Primer 0.05 5 0.5 µM
100µM Rvs Primer 0.05 5 0.5 µM
Aliquot 15 µL of master mix to each well.
Add 5 µL of 1/20 diluted bacterial culture to aliquoted master mix.
Total (µL) 10 20



2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 98˚C 10 min Initial denaturation.
2 98˚C 30 sec Denaturation.
3 61˚C 30 sec Annealing.
4 72˚C 1 min Extension. 15sec/kbp, but using longer
5 GOTO 2 x 29
6 72˚C 5 min Final extension.
7 4˚C FOREVER Stop reaction.


Dan Yamamoto-Evans 2019/11/25 14:48 Print at 96%

  • interactome/experiments/20191127-2.txt
  • Last modified: 2019/11/27 14:48
  • by dan