5x HOT FIREPol® MultiPlex Mix Ready To Load

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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 master mix for high throughput applications with ready-to-load feature

  • analyze multiple targets per reaction
  • increased sensitivity and yield
  • prevents primer dimer formation
  • reaction set-up and shipment without dry ice

Ordering

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Catalog number

04-36-00S20

Quantity

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Details

Overview

Description

5x HOT FIREPol® MultiPlex Mix Ready to Load  is a premixed ready-to-use solution containing all reagents required for hot-start multiplex PCR (except template, primers and water) and an additional compound needed for direct loading onto agarose gel and two tracking dyes (blue and yellow) that allow to monitor progress during electrophoresis.

Generated PCR products are compatible with T/A cloning procedures.

Recommendations

We recommend using HOT FIREPol® MultiPlex Mix Ready To Load in any PCR application that will be visualized by agarose gel electrophoresis and ethidium bromide staining.

HOT FIREPol® MultiPlex Mix Ready To Load is not recommended for use in applications where spectro-photometric measurements (absorbance or fluorescence) are necessary because yellow and blue dyes can interfere with these applications.

Properties

Concentration: 5x

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

Ready to load: yes

Fidelity: 1 x Taq

Cloning Type: T/A cloning 

Multiplexing: tested with up to 18 targets (depending on amplicon length)

Amplicon Size: up to 5 kb

Mix Components

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

5x Multiplex Buffer with 10 mM MgCl21x PCR solution – 2 mM MgCl2

dNTPs: dATP, dCTP, dGTP and dTTP

Bovine serum albumine (BSA) to enhance PCR reaction

Blue dye: migration equivalent to 3.5-4.5 kb DNA fragment

Yellow dye: migration equivalent to 35-45 bp DNA fragment

Compound that increases sample density for direct loading 

Sensitive and specific results
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.
After first-strand cDNA synthesis, how much cDNA template should I use for my PCR?

Recommended final amount of cDNA sample in downstream PCR reaction is up to one tenth of the final reaction volume. Overload of cDNA sample may compromise the downstream PCR, because cDNA sample may contain reaction components that may inhibit your PCR reaction.

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 HOT FIREPol® Multiplex Mix Ready to Load

Shipping

At room temperature