Barcoded cDNA generation with fusion PCR.

Protocol

1 Reaction preparation

List of paramater tested.

RT + + Memo _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Tm/Target gene MORF4L1 MRFAP1
Row/Col on PCR plate 1 2
Frd primer PI 11 PI 13
Rvs Primer PI 2 PI 2
Row/Template
A 7-20 5-20
B 7-30 5-30
C 7-40 5-40
D 7-50 5-50
E 8-20 6-20
F 8-30 6-30
G 8-40 6-40
H 8-50 6-50



  • 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 6.12 306 -
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 17 µL to each tube
10µM Frd Primer 0.5 1 0.5 µM
10µM Rvs Primer 0.5 1 0.5 µM
Fusion PCR template 0.5 1
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. 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 2019/12/10 11:37 Print at 96%

  • interactome/experiments/20191210-2.txt
  • Last modified: 2019/12/10 11:54
  • by dan