Michael Court
Updated September 2006
Basic PCR technique
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Protocol based on Platinum Taq DNA Polymerase
Wear gloves, adhere to Rnase free protocols, and use filter tips
Can do at room temperature if use Platinum Taq (any "Hot-Start" type)
Individual component (homebrew)method:
In PCR tube (25uL recipe):
a. Add balance water to 25 uL final volume
b.
c.
d.
e.
f.
g.
h.
Add 0.5 uL dNTP mix (l0 mM each)
Add 1 uL MgCl2 (50 mM)
Add 2.5 uL 10X PCR buffer
Add 1 uL EACH of forward and reverse primers (5 pmoles / uL; 5 uM)
Mix by pipetting up and down or flicking tube
Brief centrifuge if needed to collect liquid at bottom of tube
Add 0.25 uL platinum Taq DNA Polymerase
i. Mix by pipetting up and down with the same pipette tip
i. Add 0.5 uL template (cDNA or 50 ng genomic DNA)
i. ***Always try to add template last to decrease the chance of cross
contamination
ii. Mix by pipetting up and down with the same pipette tip
j. NOTE: For multiple tubes (>5) it is easier if you make up a master mix (i.e. all
ingredients combined together except template), which you add to each tube (minus
template), followed by the template. In this case use only 0.25 uL Taq per 25 uL
volume.
Commercial master mix method (e.g. Invitrogen Platinum supermix):
In PCR tube (25uL recipe):
a.
b.
c.
d.
Add 22.5 uL of 1.1x Supermix (contains Platinum taq, dNTPs, MgCl and buffers)
Add 1 uL EACH of forward and reverse primers (5 pmoles / uL; 5 uM)
Add 0.5 uL template (cDNA or 50 ng genomic DNA)
Mix by pipetting up and down
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For regular PCR:
a. Place in PCR machine and perform 35 to 40 cycles of:
b. 950C for 10 min --- 1 cycle
c. 950C for 30 sec; “anneal” temperature (about 3 to 5 degrees above primer melting
temperature – verify by “gradient” PCR) for 30 sec; 720C for 1 minute per 1 – 2 kb
of expected product length --- 35 to 40 cycles
d. 720C for 10 minutes
e. 250C for 1 sec
f. Stop
For “Gradient” PCR:
a. Place in PCR machine and perform 35 to 40 cycles of:
b. 950C for 10 min --- 1 cycle
c. 950C for 30 sec; gradient of 560C to 720C for 30 sec; 720C for 1 minute per 1 – 2 kb
of expected product length --- 35 to 40 cycles
d. 720C for 10 minutes
e. 250C for 1 sec
f. Stop
For “Touchdown” PCR:
a. Place in PCR machine and perform 35 to 40 cycles of:
b. 950C for 10 min --- 1 cycle
c. 950C for 30 sec; 650C for 30 sec; 720C for 1 minute per 1 – 2 kb of expected product
length --- 20 cycles with anneal temperature decreasing from 650C to 450C by 10C
per cycle (20 X 10C = 200C)
d. 950C for 30 sec; 450C for 30 sec; 720C for 1 minute per 1 – 2 kb of expected product
length --- 15 to 20 cycles
e. 720C for 10 minutes
f. 250C for 1 sec
g. Stop
8.
Analyze 10 uL on DNA agarose gel