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:

 

Vol.

Final conc.

10X Pfx amplification buffer

5 ml

1X

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 *

 

ddw

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

Annealing

30 sec  - 55˚C

---------

Extension

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.