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