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Some applications of this product  may require a license which is not provided by the purchase of this product.

For research use only. 

Hot-start PCR mix for GC-rich templates

  • excellent amplification with templates up to 79% GC content
  • suitable for templates up to 5 kb
  • suitable for fragment analysis

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Catalog number

04-33-00115

Quantity

€ 40.00

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Details

Reagents Provided
Item Pcs. Vial size
5x HOT FIREPol® GC Master Mix 1 1 ml | 250 rxn
25 mM MgCl2 1 0.5 ml
DMSO 100% 1 0.5 ml
Overview

Description

HOT FIREPol® GC Master Mix x that has been developed for working with difficult GC-rich templates and DNA secondary structures. It shows excellent amplification with templates up to 79% GC content

The master mix contains hot-start Taq polymerase HOT FIREPol®, MgCl2, dNTPs and a special buffer for high yield GC-rich amplification.

Extra vials of 100% DMSO and 25 mM MgCl2 enable flexibility in reaction optimization. 

Properties

Concentration: 5x

Hot-start: yes, initial activation in 12-15 min.

Ready to load: no

Fidelity: 1 x Taq

Cloning Type: T/A cloning

Amplicon Size: up to 5 kb

Difficult templates: robust on GC-rich templates

Mix Composition:

HOT FIREPol® DNA polymerase: chemically modified FIREPol® DNA Polymerase enabeling hot-start

5x HOT FIREPol® GC Buffer with 7.5 mM MgCl21x PCR solution –1.5 mM MgCl2

dNTPs: dATP, dCTP, dGTP and dTTP

Bovine serum albumine (BSA) to enhance PCR reaction

In separate vials:

100% DMSO is included in the kit in a separate vial. DMSO is recommended as a PCR additive for templates with high GC content. In some cases DMSO is also required to relax secondary structures.

25 mM MgClfor further optimization

Amplicons of various GC-content
Amplicons of various lengths from GC-rich template
Related Resources

FAQ

What is the function of BSA in Master Mix?

Bovine serum albumine (BSA) is one of the PCR additives that is used to enhance PCR reaction.

It has been proved that BSA increases PCR yields from low purity templates containing iron chloride, hemin, fulminic acid, humic acid, tannic acid, stool extracts and melanin (Al-Soud and Rådström 2000; Scipioni et al. 2008b; Opel et al. 2010).

Moreover, BSA also improves specificity in amplification of regions with secondary structures. It is also reported to prevent reaction components from sticking to tube walls.

Which polymerases / master mixes are compatible with a downstream restriction enzyme digest without cleaning up the PCR reaction?

FIREPol® and HOT FIREPol® DNA Polymerases and Master Mixes, as well as SolisFAST® Master Mix are compatible with most restriction enzymes without cleaning up the PCR reaction product.

SolisFAST® Master Mix with UNG contains dUTP instead of dTTP leading to the generation of PCR products containing uracil. Check your restriction enzyme for the ability to digest U-containing DNA amplicons.

Which DNA polymerases / master mixes should I use to obtain PCR product suitable for Sanger sequencing?

Products generated by our FIREPol®, HOT FIREPol® DNA Polymerases and Master Mixes, as well as SolisFast® Master Mixes may be used for Sanger sequencing.

Prior to the sequencing, the products should be purified to remove excess primers and nucleotides (enzymatically with ExoI/SAP or Spin column-based nucleic acid purification).

Products generated with 5x HOT FIREPol® Blend Master Mix should be cleaned up utilising column-based nucleic acid purification method, because this mix contains proofreading enzyme with 3’-5’ exonuclease activity and may degrade your sequencing primer.

Please note that Ready to Load versions of our Master Mixes are not recommended for use in applications where spectro-photometric measurements (absorbance or fluorescence) are necessary because tracking dyes can interfere with these applications.

Can I use Solis BioDyne polymerases / master mixes for colony PCR?

Yes, Solis BioDyne HOT FIREPol® DNA Polymerase and 5x HOT FIREPol® Blend Master Mix are suitable for colony PCR to determine the presence or absence of insert DNA in plasmid constructs directly from bacterial colonies without culturing or plasmid purification steps.

Sometimes, poor quality of the colony material (e.g. presence of contaminants) could inhibit the PCR reaction. It is important to use freshly grown colonies (overnight growth). If possible, include a positive (e.g. purified plasmid DNA with a desired insert) and a negative control (containing all components except template DNA) in your experimental set-up.

What would be the recommended PCR Master Mix for GC-rich samples?
GC-rich DNA is difficult to amplify, because it forms stable secondary structures and may cause unspecific amplification. Solis BioDyne 5x HOT FIREPol® GC Master Mix works very well with GC-rich samples demonstrating excellent amplification of regions with GC content up to 79%, and can amplify a fragment up to 5 kb. We would suggest to start with the recommended protocol. 5x HOT FIREPol® GC Master Mix is supplied with 100% DMSO and 25mM MgCl2 enabling further optimization of the reaction conditions.
What are the storage conditions and stability of Solis BioDyne reagents?

Solis BioDyne products should be stored at -20°C.

Shipping and temporary storage for up to 1 month at room temperature (*1525°C) has no detrimental effects on the quality of Solis BioDyne reagents.

Freeze-thaw stability is tested for each product. Most PCR and qPCR products have passed 30 freeze-thaw cycles with no changes in performance. Specific information is found in Storage and Shipping conditions of each product.

When stored and handled under the recommended conditions, full activity of the reagents is retained until the Expiry Date printed on the tube label.

*World Health Organization (2003). Guidelines for the Storage of Essential Medicines and Other Health Commodities.

Storage & Shipping
Storage

Routine storage: -20ºC

Temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of 5x HOT FIREPol® GC Master Mix

Shipping

At room temperature