Material

  • Sample : DNA template
  • Primers
  • dNTPs
  • Phusion buffer
  • Phusion pol

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


Protocol

1 Reaction preparation
  • Mix the following reagents in one tube (ideally from top to bottom)
Check Reagent Volume (µl) 1 reaction Volume (µl) _ _ _ reaction Final concenration Memo
ddH20 6.62 -
Phusion HF buffer (5x) 2.0 -
25mM dNTPs 0.08 0.2 mM Equal volume mix of 100mM dA/T/G/CTPs will give this solution.
Frd Primer 0.05 0.5 µM
Rvs Primer 0.05 0.5 µM
Transfer the tube on ice
Phusion pol 0.2 - Home brew Phusion pol
Template 1 - 1ng is enough for template.
Total (µL) 10



2 Reaction
  • Run the following program on a thermal cycler
STEP Action Time Memo
1 98˚C 30 sec Initial denaturation.
2 98˚C 10 sec Denaturation.
3 65˚C 10 sec Annealing. Depending on primer Tm
4 72˚C 15 sec Extension. Depends on amplicon length.
5 GOTO 2 x 34
6 72˚C 5:00 Final extension.
7 4˚C FOREVER Stop reaction.



3 Agarose gel electrophioresis
  • Make agarose gel
Reagent Amount Memo
Agarose powder 1.0 g This is for 1.0%. For “1kbp> Expected >500bp”, use 1.5%. For “500bp>Expected”, use 2.0%.
1x TBE buffer 100 mL
Microwave and dissolve agarose completely.
Midori green advance 10 µL DNA stain
Swirl to mix the stain uniformly, and cast gels. 100 mL will give you 2 x wide + 2 x small on heat-tolerant trays.
Wait for gel to settle.
  • interactome/pcr.txt
  • Last modified: 2019/11/22 14:31
  • by dan