Universal protocol for PCR.

No optimization required. Multiplexing also allowed.

Scalable from 10 to 50 µL.

Working on tubes, strips, plates on every thermal-cycler.

DNA

Buffer BD 10X:        

dNTPs (@ 2 mM):         

MgCl₂ (25mM):          

Primer F (10 pmol/µl):

Primer R (10 pmol/µl):

2 µL

2 µL

2.4 µL (final concentration: 3 mM)

1 µL

1 µL

Recommended use for best results: 

HOT FIRE DNA POL (5U/µL):

0.2 µL

H₂O

Thermocycler:

                                                                       95º C   15’       min

                                   20 cycles:                   95º C   1          min

                                                                       68º C   30”       sec - 1° C/cycle

                                                                       72º C   1.5”      min

                                   20 or 30 cycles:          95º C   1          min

                                                                       51º C   30”       sec

                                                                       72º C   1.5       min

                                                                       72º C   10’       min

                                                                       4º C                 forever

Un example of various genotypes from a 4-plex including genes: TP53 (intron 3 16bp ins/del polymorphism), GSTT1 (+ or null genotype) and GSTM1 (positive and null genotype). High molecular weight CYP2A6 was used as positive control.

Details on the protocol are given in this PDF.

If your PCR does not work, simply re-design the primers.

Do not spend time in adjusting the conditions.

High-specificity. Save time.

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