PCR - PLATINUM Pfx DNA Polymerase (Invitrogen)

Polymerase Description (PDF)

        Proofreading 3' à 5'  exonuclease activity

        Fast chain extension capability

        One unit of Pfx  pol incorporates 10 nmol of dNTP's in 30 min at 74˚C.

        1 ml Pfx = 2.5 units


The Pfx  pol is in an inactive form that provides "Hot start" for PCR.

Activation - denaturation step in PCR cycling at 94˚C.


For problematic and/or GC-rich templates:

          Use the PCRx enhancer solution.

          The PCRx lowers the DNA melting temp (Tm), the max primer annealing temp is lowered ~2˚C per 1X PCRx solution concentration.

          If no thermal cycling protocol is optimal for the template ==> annealing temp 55-60˚C.

          For templates with 60-90% GC content - test of 10X PCRx solution at final
concentration: 0.5X ; 1X ; 2X ; 3X


PCR protocol

1. On ice:



Final conc.

10X Pfx amplification buffer

5 ml


10 mM dNTP's mix

1.5 ml

0.3 mM each

50 mM MgSO4

1 ml

1 mM

Primer mix (10 mM each)

1.25 ml

0.3 mM each

Template DNA (10pg - 200ng)

1 ml


Pfx DNA polymerase

0.5 ml *



Up to 50 ml


* When amplifying longer targets - above 3 Kb use up to 1 ml of enzyme


2. Mix by tapping the tube.

3. Spin down briefly.

4. Denaturation of the template 2 min at 94˚C

5. PCR cycling (25-30 times)



3 step cycle

2 step cycle **

Denaturation !

15 sec -  94˚C

15 sec 94˚C


30 sec  - 55˚C



1 min per Kb - 68˚C

1 min per Kb - 68˚C

** 2 step cycle is used for primers with high Tm.

! Denaturation conditions also depend on the TM. Long primers with high TM require longer denaturation time (~45). Primers with secondary structures may require up to 2min denaturation.

6. Maintain reaction at 4˚C.

7. Store samples at -20˚C.