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