Polymerase Chain Reaction (PCR) Protocol

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> Polymerase Chain Reaction (PCR) Protocol

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 (dont 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

Turn on machine 5 minutes prior to the run
Start running the program
Wait for lid to reach maximum temperature
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 Program
Step 1: 2 min. 95C
Step 2: 1 min. 95C
Step 3: 1 min. average of melting temperature of annealing portion of 2 primers 
Step 4: 1 min. 72C
Step 5: Goto Step 2 4x
Step 6: 1 min. 95C
Step 7: 1 min. average of melting temperature of entire length of 2 primers
Step 8: 1 min. 72C
Step 9: Go to Step 6 34x
Step 10: 5 min. 72C
Step 11: forever 4C

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

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