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 (T_m), 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 T_m.

! 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.