Amplifying barcoded fragments.


Protocol

1 Reaction preparation

List of paramaters tested.

Frd primer overlap 20 30 40 50
BC type Template Rvs primer Row/Col on PCR plate 1 2 3 4 5 6 7 8 9 10 11 12
DB PI-05 PI-02 A
DB PI-06 PI-02 B
AD PI-07 PI-02 C
AD PI-08 PI-02 D
  • Mix the following reagents in one tube (ideally from top to bottom). Prepare 8 of this mix.
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.
Template DNA 1 50 1 ng/µL DNA from TWIST.
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 30 sec Initial denaturation.
2 98˚C 30 sec Denaturation.
3 65˚C 30 sec Annealing.
4 72˚C 30 sec 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/20191129-4.txt
  • Last modified: 2019/12/11 12:50
  • by dan