Polymerase Chain Reaction (PCR) Protocol

Primer can be stored in the refrigerator 4 degrees if not suspended in water
when resupending in water, using sterile water
when uncapping the tube, do it gently to prevent the spread of lyophilized primer
add enough water to have 100pmol / uL OR 1UG / uL
The primary stock dilute to 10 pmol / uL OR 100ng / uL to obtain a working solution.

Polymerase Chain Reaction (PCR) Protocol

Include a negative control DNA template is replaced with water (perhaps using a master mix)

To thin-wall 0.5ml PCR tubes (do not get this mixed up with another 0.5ml tubes),
added in this order:
DNA template 5uL
water up to 50uL
10x polymerase buffer 5uL
0.5uL dNTP (a mixture of equal amounts the example 1.25uL)
Primer 1 1UL of 10 pmol / uL
Primer 2 1UL of 10 pmol / uL

Turn the engine 5 minutes before the run
Start the program
Wait close to reaching the maximum temperature
Adding enzymes 1UL sample, pipette up and down 6x, mixed with finger
Press pause again to start the program
(Please note that each machine may differ in the way they should be operated.)

The PCR program
Step 1: 2 minutes. 95C
Step 2: 1 min. 95C
Step 3: 1 min. The average temperature of the melting portion of the second primer annealing
Step 4: 1 min. 72C
Step 5: Goto Step 2 4x
Step 6: 1 min. 95C
Step 7: 1 min. Average temperatures melting the entire length of two primary
Step 8: 1 min. 72C
Step 9: Go to Step 6 34x
Step 10: 5 min. 72C
Step 11: forever 4C

Polymerase Chain Reaction (PCR) Protocol

if the melting temperature of both the primary the same, no need to divide the program into the second round of the cycle

running the PCR products on a gel and excise out and purify the band right before digesting Alternatively, PCR can be purified by PCR purification kit if you are busy with something else