Re-trying with Petr's protocol.
Step Description Date/Initials Link protocol Link results
1 Total RNA extraction from HEK293T cells 20191122DEY
2 1st strand cDNA synthesis 20191122DEY
3 Target gene amplification from cDNA sample Current
4 BP-reaction
5 Sanger sequence check


Protocol

1 Reaction preparation

List of paramater tested.

RT - + - + - + - + - +
Tm/Target gene MORF4L1 MORF4L1 MRFAP1 MRFAP1 BCL2L2 BCL2L2 BIK BIK MRFAP1L1 MRFAP1L1
Row/Col on PCR plate 1 2 3 4 5 6 7 8 9 10
  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) 1 reaction Volume (µl) for 25 reactions with 20µL vol Final concenration Memo
ddH20 4.92 246 -
Phusion HF buffer (5x) 2.0 100 -
25mM dNTPs 0.08 4 0.2 mM Equal volume mix of 100mM dA/T/G/CTPs will give this solution.
DMSO 0.8 40
cDNA template 1 50 Diluted cDNA sample.
Transfer the tube on ice
Phusion pol 0.2 10 - Home brew Phusion pol
Aliquot 18 µL to each tube
10µM Frd Primer 0.5 1 0.5 µM
10µM Rvs Primer 0.5 1 0.5 µM
Total (µL) 10 20



2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 98˚C 3 min Initial denaturation.
2 98˚C 10 sec Denaturation.
3 65˚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/20191125-3.txt
  • Last modified: 2019/11/25 20:40
  • by dan