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MBI EVOlution 5 x EvaGreen® HRM Mix (No Rox) (1000 Reactions)

Catalog Number:
MBI-HRM1000
Availability: Out of stock
$825.00

For in vitro use only

Description:

MBI EVOlution 5 x EvaGreen® HRM Mix (no ROX) is an optimised ready-to-use solution for High Resolution Melt (HRM) Analysis, incorporating EvaGreen® dye. It comprises all the components necessary to perform qPCR and HRM Analysis: MBI Hotstart DNA Polymerase, ultrapure dNTPs, MgCl2 and EvaGreen® dye. The user simply needs to add water, template and primers.

MBI Hotstart DNA Polymerase is activated by a 15 min incubation step at 95°C. This prevents extension of non-specifically annealed primers and primer-dimers formed at low temperatures during qPCR setup.

 

Applications:

  • High Resolution Melt (HRM) 

 

Mix Composition:

  • MBI Hotstart DNA Polymerase
  • 5 x EvaGreen® HRM buffer
  • 12.5 mM MgCl2

1 x PCR solution – 2.5 mM MgCl2

  • dNTPs, including dTTP to improve reaction sensitivity and efficiency compared to dUTP
  • EvaGreen® dye
  • BSA 

 

EvaGreen® Dye:

EvaGreen® is a DNA-binding dye with many features that make it a superior for HRM. Apart from having similar spectra, EvaGreen® has three important features that set it apart from SYBR® Green I: EvaGreen® has much less PCR inhibition, is extremely stable dye and has been shown to be nonmutagenic and noncytotoxic. EvaGreen® is compatible with all common real-time PCR cyclers – simply select the standard settings for SYBR® Green or FAM!

 

Recommended qPCR reaction mix:

Component

Volume

Final conc.

MBI EVOlution 5 x EvaGreen® HRM Mix (no ROX)

4 µl

1x

Primer Forward

(10 pmol/µl)

0.16-0.5 µl

80-250 nM

Primer Reverse

(10 pmol/µl)

0.16-0.5 µl

80-250 nM

DNA template

1-5 µl

1-50 ng/µl

H2O PCR grade

up to 20 µl

 

Total

20 µl

 

 

Safety warnings and precautions:

This product and its components should be handled only by persons trained in laboratory techniques. It is advisable to wear suitable protective clothing, such as laboratory overalls, gloves and safety glasses. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water.

EvaGreen® is a registered trademark of BIOTIUM, INC.

The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis.  The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes.

SYBR® is a registered trademark of Molecular Probes, Inc. LightCycler®  is a registered trademark of Roche Molecular Systems, Inc

Some applications this product is used in may require a license which is not provided by the purchase of this product. Users should obtain the license if required.

 

Recommended cycles for HRM on LightCycler® 480 System

Target (°C)

Acquisition Mode

Hold (mm:ss)

Ramp Rate (°C/s)

(96 –well/384-well)

Acquisitons (per °C)

Initial denaturation

95°C

None

15:00

4.4/4.8

-

Amplification

95°C

None

00:10

4.4/4.8

-

Primer dependent 1)

None

00:15

2.2/2.5

-

72°C

Single

00:10-00:30 2)

4.4/4.8

-

High Resolution Melting

95°C

None

01:00

4.4/4.8

-

40°C 3)

None

01:00

2.2/2.5

-

65°C 4)

None

00:01

1/1

25

95°C 4)

Continuous

-

-

 

Cooling

40°C

None

00:10

2.2/2.5

-

In case you do not know the melting temperatures of your PCR primers exactly, it is recommended to apply a touchdown PCR protocol covering a range of annealing temperature from 65 to 53°C.

  1. Annealing temperature is the parameter that most influences specificity and robustness of amplification. For initial experiments set the target temperature 2°C below the calculated primer Tm. The amount of specific product, the presence/absence of undesirable side product, and the presence/absence of dimer product in these experiments will dictate the best way to optimize this parameter. If the reaction produces undesirable product, increase the annealing temperature. If amplification is not robust, decrease the annealing temperature and/or increase the duration of the annealing step.
  2. Calculate the exact elongation time required for your specific target by dividing the amplicon length by 15 (e.g., a 450 bp amplicon requires 30 s elongation time).
  3. This pre-hold temperature ensures that all PCR products have re-associated and encourages heteroduplex formation.
  4. Actual melting conditions depend upon the amplicon. For initial experiments set a wide melting interval, e.g., from 60 to 95°C. Once you have determined where the product will melt, reduce the melting interval to approximately 25°C. Ensure that the melt program starts at least 10°C before and ends at least 10°C after the expected Tm value.

 

Shipping and Storage conditions:

Routine storage: -20ºC

Shipping and temporary storage for up to 1 month at room temperature has no detrimental effects on the quality of MBI EVOlution 5 x EvaGreen® HRM Mix (no ROX).

More Information
Amount of Reactions 1000 Reactions
Mix Composition No ROX Dye
Application HRM Evagreen
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