5× Medino® PCR Master Mix(for multiplex PCR)

Product5× Medino® PCR Master Mix(for multiplex PCR)
Catalog No.LM201012-2
Storage conditionsThis product should be stored at -20℃ for 2 years.

Description

Product description

5× Medino® PCR Master Mix (for Multiplex PCR) is developed based on a new generation of high-fidelity DNA, polymerase Canace, and is specifically optimized for multiplex PCR library construction applications in the field of high-throughput sequencing. The Canace enzyme is based on Pyrococcus furiosis DNA Polymerase and is modified by genetic engineering. The protein structure contains the 5 ′ 3 ′ polymerase domain and the modified 3 ′ 5 ′ exonuclease domain. This combination greatly improves the heat resistance and fidelity of the enzyme. It is 98℃, 52 times more fidelity than Taq DNA polymerase and 6 times more fidelity than common Pfu DNA polymerase. Unique elongation factors were added to the enzyme solution and the amplification was up to 15 sec / kb. This product is a 5 mix PCR mixture, with a large volume range of added templates and primers, making your experiment more flexible (our company has another 2 mix available).

 

Components

Cat.No.LM201012-2
Size1 mL/500mL

 

Instructions


  1. Recommended PCR reaction systems.
ComponentsVolume (μL)Final concentration
5× Medino® PCR Master Mix (for multiplex PCR)10
Templatex
Primer mix (X μM)x
ddH2OUp to 50

[Note]:

  1. a) Please mix each components before use.
  2. b) Primers and template: The first round of multiplex PCR for target enrichment, in which the primers    and template conditions need to be explored. The concentration of the second round of multiplex           PCR is recommended: 10 μM primer plus about 2 μL.

 

  1. Reaction program

First-round PCR amplification procedure:

Cycle stepTemp.TimeCycles
Initial denaturation98℃3min1
Denaturation98℃20 sec

 

15-45

Annealing60℃4-10 min
Extension60℃4-10 min
Final extension72℃5 min1

[Note]:

  1. Set the reaction procedure in the above table on the PCR instrument to perform the multiple PCR reaction (set the hot cover temperature to about 105℃).
  2. Annealing / extension time: 100 heavy PCR recommended 4 min; 1000 heavy recommended 8-10 min according to the primer situation of exploration.

The PCR products were purified by magnetic beads

It is recommended to add 45 μ L magnetic beads to the 50 μ L reaction system for product purification

 

Second-round PCR amplification procedure:

Cycle stepTemp.TimeCycles
Initial denaturation98℃30 sec1
Denaturation98℃15 sec

 

25-35

Annealing60℃30 sec
Extension72℃1min
Final extension72℃5 min1

[Note]:

  1. Annealing temperature and time: 60℃ is recommended, and a temperature gradient can also be set     up to explore the optimal temperature of primer annealing as needed. The recommended annealing               time is set to 30 sec, which can be adjusted within 10-30 sec. Too long an annealing time may cause          the dispersion of the amplification product on the glue.

 

The PCR products were purified by magnetic beads

It is recommended to add 45 μ L magnetic beads  to the 50 μ L reaction system for product purification

Notes

  1. a) For your safety and health, please wear a lab coat and disposable gloves during operation.
  2. b) This product is for research use only!
  3. c) It is worth noting that the multiple PCR library building experiment itself is difficult, which usually needs to be optimized. If you encounter difficulties when using this product, you can contact our               technical support. We have multiple optimization schemes, which can help you to better build the     experimental system.

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