Volcano2G DNA polymerase

Product#: 8100
$144.00

Size:

  • 250 Units
  • 1000 Units
Availability:
Ships in 1-2 weeks

Volcano2G DNA polymerase

Cat. No. 8100
Size 250 units / 1000 units
Store -20°C, This product is shipped on cool packs. It is recommended to store the product upon arrival at -20°C.


Description

Volcano2G DNA polymerase is an engineered, extremely thermostable reverse transcriptase and combined DNA polymerase, obtained through directed, artificial evolution. Volcano has a half-life at 95°C of >40 min. The Volcano2G reverse transcriptase and DNA polymerase facilitates “zero-step” RT-PCRs directly from RNA templates (without an isothermal reverse transcription step), as reverse transcription takes place simultaneously with DNA amplification during the cycled PCR elongation step. This also allows reverse transcription reactions at high temperatures, thus minimizing the problems encountered with strong secondary structures in RNA that melt at elevated temperatures.

Recommendations for PCR/ Reaction Setup
PCR Mix

Component

Volume

Final concentration

Primer forward (10 µM)

1 µl

0.5 µM (50-1000 nM)

Primer reverse (10 µM)

1 µl

0.5 µM (50-1000 nM)

dNTPs (10mM)

2 µl

400 µM

5x Volcano buffer

10 µl

1x

Volcano2G DNA polymerase (5 U/µl)

1 µl 5

Units/reaction

Template/Sample extract*

various

>1 ng (1-1000 ng)

Nuclease-free

water up to 50 µl total reaction volume

Keep all components on ice. Spin down and mix all solutions carefully before use.
* Recommended final template concentration is between 5-500 ng/µl.

Content
Volcano2G DNA polymerase is supplied as a 5 U/µl solution containing glycerol. It comes together with an 5x optimized reaction buffer. Hot-start formulation of Volcano2G DNA polymerase prevents false amplification during the reaction set-up.

Quality Control
RT-PCR activity: Volcano2G is tested for a successful RT-PCR performance. A 151 bp fragment (HPRT1 mRNA) is amplified from human total RNA extract in a PCR cycler and visualized as a single amplified product. DNA polymerase activity: Volcano2G polymerase activity is monitored and adjusted to a specific DNA polymerase activity using an artificial DNA template and DNA primer. Enzyme-concentration is determined by protein-specific staining. Please inquire more information at info@mypols.de for the lot-specific concentration.

Safety
This product does not require a Material Safety Data Sheet because it does neither contain more than 1% of a component classified as dangerous or hazardous nor more than 0.1% of a component classified as carcinogenic. However, we generally recommend the use of gloves, lab coats and eye protection when working with these or any other chemical reagents. myPOLS Biotec takes no liability for damage resulting from handling or contact with this product. Further information can be found in the REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL.

Typical 0-step RT-PCR protocol
(an isothermal reverse transcription step is not needed)

Initial denaturation

95°C

2 min

Denaturation

95°C

15 sec

Annealing/Extension*

Various

45 sec (25-40 cycles)

Optional melting step

 

 

A two-step as well as three-step PCR protocols can be used.
* A new RT-PCR is ideally established by running a temperature gradient in order to find the best annealing / extension temperature for each primer pair. The annealing temperature of a primer is influenced by its nucleic acid sequence and the reaction buffer composition (salts and pH). Volcano2G DNA polymerase is most active between 50-95°C

 
Important notes
  • Volcano2G DNA polymerase is engineered and optimized for an amplicon size between 60- 300 bp.
  • Volcano2G DNA polymerase can also be used for real-time cycling, when adding a suitable real-time PCR dye or a fluorescent probe.
  • Volcano2G DNA polymerase does not require Mn2+ for optimal reverse transcription activity. However, some assays may be further optimized by the addition of Mn2+ to the reaction (suggested concentration is 0.5 - 1 mM).
Licences/Patents/Disclaimers
This product is covered by a pending patent application. It is for the purchaser’s own internal research use and may not be resold, modified or used for production and commercial purposes of any kind without an agreement with myPOLS Biotec. For information on obtaining additional rights, please contact: info@mypols.de. The product is for research use only and may be used for invitro experiments only.

References
  1. Volcano2G DNA polymerase is based on: 2. Structure and Function of an RNA-Reading Thermostable DNA Polymerase. Angew. Chem. Int. Ed., 2013; 52: 11935–11939. Blatter, N., Bergen, K., Nolte, O., Welte, W., Diederichs, K., Mayer, J., Wieland, M. and Marx, A.
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