Standard PCR protocol

Reagents:

  1. Water (Nuclease-free)
  2. 10x PCR buffer*
  3. MgCl 2 *
  4. Taq polymerase*
  5. dNTP (25 mM each)**
  6. Primers (5uM each)***

Notes:

* Comes with Qiagen HotStarTaq DNA Polymerase kit

**Mix equal volumes of 100 mM stocks of Invitrogen dATP, dTTP, dCTP and dGTP

***Primer stocks are at 100 uM; Add 95 ul water for every 5 ul primer to obtain 5 uM solution

Reaction recipe (10 ul volume):

  1. 4.7 ul water
  2. 1.25 ul 10x PCR buffer
  3. 0.65 ul MgCl 2
  4. 0.20 ul dNTP (25 mM each)
  5. 1.0 ul primer 1
  6. 1.0 ul primer 2
  7. 0.2 ul Taq Polymerase

Notes:

  • Use 1 ul template DNA or cDNA; quick spin templates to the bottom of the tubes
  • Make a master mix with the appropriate volumes of each reagent, according to the number of reactions
  • Add 9 ul master mix to each template; pipet up and down to mix; quick spin samples to bottom of tubes
  • Perform thermal cycling; start with 95C for 15 min to activate Taq; adjust annealing temp according to primers