PCR Protocol for Screening
1. Sign up for a PCR machine and make sure your program is ready.
Basic Program:
Heat lid at 105°C without preheating if available
1 X 95°C for 1 min
34 X 95°C for 30s
65°C for 1 min (temp may vary with primers)
72°C 1 min per kb
1 X 72°C for 10 min
4°C hold
2. Prepare the Master Mix on ice without the Taq polymerase. Prepare for more
reactions than you need (~ 1.1 X more usually works).
Master Mix 1 (10+ samples, 25μL final volume)
X 1 rxn
X 10
ddH20
18.6μL
186μL
10X PCR Buffer
2.5
25
2.5 mM dNTPs
2.5
25
Primer 1, 100μM
0.1
1
Primer 2, 100μM
0.1
1
Taq, lab stock
0.2
2
X 20
372μL
50
50
2
2
4
Master Mix 2 (1 - 10 samples, 25μL final volume)
X 1 rxn
X5
ddH20
17.8 μL
89.0 μL
10X PCR Buffer
2.5
12.5
2.5 mM dNTPs
2.5
12.5
Primers 1 & 2, 10μM both* 1.0
5.0
Taq, lab stock
0.2
1.0
X 10
178 μL
25
25
10
2
*this is a 1:10 dilution of both 100μM primer stocks together
3. Spot 1 μL DNA or cDNA into each sample well on ice.
4. Add the Taq to the Master Mix. Invert gently, then spin quickly.
5. Add 24 μL Master Mix to each sample directly (this should mix in the DNA at the
bottom of the tube). Use a new pipet tip for each sample.
6. Overlay samples with 2 drops of mineral oil.
7. Place plastic sheet over PCR plate.
8. Put samples in PCR machine and start program.