PCR Protocol with a Thermal Cycler

Biochemistry, Molecular Biology, and Cell Biology Protocols >> PCR Protocol with a Thermal Cycler

Prior to starting the experiment, thoroughly read the manual for the PCR thermal cycler that you will be using.

Making up primers
Primers may be kept in the 4-degree fridge if not resuspended in water
when resupending in water, use sterile water
when uncapping the tube, do it gently to prevent dispersal of the lyophilized primers
add enough water to have 100pmol/uL OR 1ug/uL
dilute the primer stock to 10pmol/uL OR 100ng/uL to obtain working solution

Include a negative control with the DNA template replaced with water (perhaps use a master mix)

Into 0.5mL thin-wall PCR tubes (don’t get this mixed up with the other 0.5mL tubes),
add in this order:
DNA template 5uL
water up to 50uL
10x polymerase buffer 5uL
dNTP 0.5uL (of a mixture of equal amounts of it e.g. 1.25uL)
Primer 1 1uL of 10pmol/uL
Primer 2 1uL of 10pmol/uL
stop

Turn on the thermal cycler 5 minutes prior to the run
Start running the program
Wait for thermal cycler lid to reach maximum temperature
Pause
Add 1uL enzyme to samples, pipet up and down 6x, mix with fingers
Press pause again to start the program
(Please note that every machine may differ in how they need to be operated.)

PCR Thermal Cycler Program
Step 1: 2 min. 95°C
Step 2: 1 min. 95°C
Step 3: 1 min. average of melting temperature of annealing portion of 2 primers
Step 4: 1 min. 72°C
Step 5: Goto Step 2 4x
Step 6: 1 min. 95°C
Step 7: 1 min. average of melting temperature of entire length of 2 primers
Step 8: 1 min. 72°C
Step 9: Go to Step 6 34x
Step 10: 5 min. 72°C
Step 11: forever 4°C

if melting temperatures of both primers are similar, it is not necessary to divide the program into 2 rounds of cycles

run the PCR product on a gel and excise out and purify the correct band before digesting
alternatively, the PCR may be purified with the PCR purification kit if you are busy with something else