Medino® Blood Advanced Direct PCR Kit

ProductMedino® Blood Advanced Direct PCR Kit
Catalog No.LM201010-1
Storage conditionsThis product should be stored at -20℃ for 1 years.

Description

Product description

Medino® Blood Advanced Direct PCR Kit is a kit for direct PCR amplification of whole blood samples without DNA purification or sample pretreatment, compatible with fresh blood containing EDTA, heparin, citrate and other conventional anticoagulant, refrigerated (frozen) blood, and commercial dry blood stains for Whatman903® and FTA®. Kit containing DNA Polymerase combinations genetically engineered, With its high fidelity and strong tolerance to PCR inhibitors,  2× Medino® Blood Advanced PCR Buffer addition of strong elongation factor, amplification enhancement factor and optimized buffer system, Ability efficient amplification of 8 kb genomic fragments under rapid extension conditions, For fragments below 2 kb, Set the 3-5 sec/kb extension time to complete the amplification, Thus greatly shorten the identification of blood samples and testing time. The 3 ′ end of the  Medino® Advanced High-Fidelity DNA Polymerase Mix is flat and suitable for one-step rapid cloning and TOPO cloning kit. The primer premix Positive control primer mix (10 μM each) provided in the kit amplified a 237 bp fragment from the conserved sequence upstream of the sox21 gene in mammals and most vertebrates and can be used as a positive control.

Components

Cat.No.LM201001-2ES01/ LM201001-2ES03/ LM201001-2ES08
Size250 μL/1 mL/5×1 mL

Instructions

  1. Recommended PCR reaction systems.
ComponentsVolume (μL)Final concentration
2×   Medino®   Blood Advanced Direct PCR Kit25
Templatex
Forward Primer 10 μmol/L2
Reverse Primer 10 μmol/L2
ddH2OUp to 50
Medino® Advanced High-Fidelity DNA Polymerase Mix1
Blood samplesX
[Note]:
  1. a) Please mix each components before use.
  2. b) The recommended final concentration of each primer is 0.4 μM, which leads to non-specific
  3. c) The optimal whole blood template concentration ranged from 0.5% to 20%, and the recommended use              was 10% as the initial attempt condition, namely 5 μL whole blood was added to the 50 μL              reaction system, with attention to avoid absorbing blood clot. For the dry blood stains stored on        the Whatman®          filter paper card, about 1 mm 2 of round paper pieces with blood stains should be   recommended, which can be amplified directly into the PCR reaction solution without pretreatment
  1. Reaction program
Cycle stepTemp.TimeCycles
Initial denaturation95℃5 sec1
Denaturation95℃15 sec30-35
Annealing60℃15 sec
Extension72℃3-10 sec/kb
Final extension72℃5 min1
[Note]:
  1. a) The annealing temperature is the universal Tm or 1 – 2℃ below the primer Tm. If the amplification              product is less specific, an annealing temperature gradient can be established to find the optimal              annealing conditions.
  2. b) The extension time of 10 sec/kb can amplify most of the target fragments below 8 kb, and most of them below 2 kb at 3-5 sec/kb. If the amplification efficiency is low, the time can be appropriately            extended to 20-30 sec/kb, and should not exceed 60 sec/kb.

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 recommended that blood template use is 10% of the total reaction volume, that is, to add 5 μL              whole blood as a template in the 50 μL reaction system, and pay attention to avoid absorbing             blood clot.
  4. d) The extension time of 10 sec/kb can amplify most of the target fragments below 8 kb, and most of them below 2 kb at 3-5 sec/kb. If the amplification efficiency is low, the time can be           appropriately extended to 30 sec/ kb.
  5. e) After the PCR reaction, it is recommended to centrifuge the reaction product at 4000 rpm (1000 g) for 1-3 min to precipitate blood cell debris, and take the supernatant for downstream analysis.

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