Generating template for barcoded cDNA generation primer from barcoded library samples.

Protocol

1 Reaction preparation

List of paramater tested.

  • 10 different barcodes (purified plasmid sample) x 2 barcode types
  • MORF4L1-40bp overlap for all genes
  • 5 pilot genes with 2 barcodes each.



  • 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.
DNA tempalte (10ng/µL) 1 50 Diluted plasmid DNA 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 30 sec 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/20191220.1576802573.txt.gz
  • Last modified: 2019/12/20 09:42
  • by dan