PCR - Bio-X-Act (from BioLine)

BIO-X-ACT is a proprietary formulation of several enzymes which delivers processivity and high specificity in long or difficult reactions. BIO-X-ACT is suitable for use on a wide range of templates at varying degrees of purity, and can be used on templates of up to 30kb. The unique composition of different enzymatic activities delivers a 17-fold increase in fidelity in comparison with regular Taq polymerase and is therefore suitable for reactions where accuracy is important. BIO-X-ACT possesses 5'-3' DNA polymerase activity and 3'-5' proof-reading activity which prevents mis-incorporations of nucleotides during a reaction.

BIO-X-ACT is supplied with its own unique buffer and also with "HiSpec Additive", which reduces smears, primer-dimers and spurious bands that are associated with difficult GC-rich and repetitive sequences.

PCR protocol

1. On ice:

	Vol.	Final conc.
10X opti-buffer	5 ul	1X
10 mM dNTP's mix	1.5 ul	0.3 mM each
50 mM MgCl2	1.5 ul	1.5 mM
Primer mix (10 mM each)	1.25 ul	0.3 uM each
Template DNA (10pg - 200ng)	1 ul
BioXact-polymerase	1ul	2.5U
ddw	Up to 50 ul

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	45 sec - 94˚C	45 sec- 94ºC
Annealing	45 sec - 60˚C	1 min per Kb -72˚C
Extension	>1 min per Kb -72˚C	1 min per Kb 72˚C

* 2 step cycle is used for long primers with high T_m.

Add final extension temp: 10min after end of cycles.

7. Maintain reaction at 4˚C.

8. Store samples at -20˚C.

If sequence is GC rich, calibrate conditions with 5x Hi-spec buffer