Re-trying with new cDNA sample.
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

Material

Before preparing: Check your primer Tm at NEB website (http://tmcalculator.neb.com/#!/main with Phusion/HF buffer setting)




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
Frd primer PI 11 PI 11 PI 13 PI 13 PI 15 PI 15 PI 17 PI 17 PI 19 PI 19
Rvs Primer PI 12 PI 12 PI 14 PI 14 PI 16 PI 16 PI 18 PI 18 PI 20 PI 20










  • 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 6.62 331 -
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.
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 X ˚C 30 sec Annealing. See Tm table
4 72˚C 3 min Extension. 15sec/kbp, but using longer
5 GOTO 2 x 29
6 72˚C 5:00 Final extension.
7 4˚C FOREVER Stop reaction.



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

  • interactome/experiments/20191210.1575944866.txt.gz
  • Last modified: 2019/12/10 11:27
  • by dan