Medino® Universal TaqMan multiplex qPCR Master Mix (UDG Based)

 

Catalog No.LM602010
Storage conditionsThe product should be stored at -25°C      -15°C for 18 months.
Hot StartBuilt-in hot start
Detection methodPrimer-probe detection
PCR methodqPCR
PolymeraseTaq DNA polymerase
Type of sampleDNA
Application equipment

 

Applied Biosystems: 5700, 7000, 7300, 7700, 7900HT Fast, StepOne™, StepOne Plus™, 7500, 7500 Fast, ViiA™7, QuantStudio™ 3 and 5, QuantStudio™ 6,7,12k Flex;

Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4;

Eppendorf: Mastercycler ep realplex, realplex 2 s;

Qiagen: Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;

Roche Applied Science: LightCycler 480, LightCycler 2.0; Lightcycler 96;

Stratagene: MX3000P™, MX3005P™, MX4000P™;

Thermo Scientific: PikoReal Cycler; Cepheid: SmartCycler; Illumina: Eco qPCR.

Product description

Medino® Universal TaqMan multiplex qPCR master mix (UDG plus) is a 2× pre-mixed reagent capable of conducting up to quadruple fluorescence quantitative PCR reactions in a single reaction well. This product contains genetically modified antibody-based hot-start Taq enzyme, greatly enhancing amplification sensitivity and specificity. Additionally, this product has undergone in-depth optimization of the multi-reaction buffer system, which can improve the amplification efficiency of reactions and promote effective amplification of low-concentration templates. This product can be used for gene typing and gene multiplex quantitative analysis. It contains UDG enzyme to effectively prevent the risk of aerosol contamination.

Components

Components No.

 

LM602010ES03/ LM602010ES08/LM602010ES10/

LM602010ES12/ LM602010ES13/ LM602010ES60/

LM602010ES62

Specifications

1 ml / 5×1 ml/ 10 ml/ 12.5 ml/

1×12.5 ml/ 20×5 ml/ 125 ml

Instructions

1.Reaction System

ComponentsVolume (μL)Final Concentration
2x TaqMan qPCR Mix12.51 x
Primer mix (10 μM)x0.1—0.5 μM
Probe mix (10 μM)x50-250 nM
Rox reference dye0.51 x
Template DNA/cDNA1—10
ddH2Oup to 25

[Note]:

  1. a) Primer concentration: Each primer pair in the Primer Mix is typically at a final concentration of 0.2 μM, which can be adjusted between 0.1-0.5 μM as needed.
  2. b) Probe concentration: Each probe in the Probe Mix, containing multiple probes with different fluorescent signals, can be adjusted between 50-250 nM depending on specific requirements.
  3. c) Rox reference dye: Used in Real-Time PCR amplifiers such as Applied Biosystems to correct fluorescence signal errors between wells; this product does not contain Rox reference dye.
  4. d) Template dilution: Due to the high sensitivity of qPCR, it is recommended to dilute the template before use. If the template is cDNA stock, the volume of template should not exceed 1/10 of the total.
  5. e) Reaction volume: It is recommended to use reaction volumes of 25 μL, 30 μL, or 50 μL to ensure effective and reproducible amplification of target genes.
  6. f) System setup: Prepare the reaction mixture in a clean workspace and use pipette tips and reaction tubes free of nucleases. It is recommended to use filter-tipped pipette tips. Avoid cross-contamination and aerosol contamination. Thoroughly mix before use to avoid excessive bubble formation.

2.Reaction program

CyclestepTemp.TimeCycles
Purification and digestion of contaminants25°C10 min1
Initial denaturation95°C5 min1
Denaturation95°C15 s

45

 

Annealing/Extension60°C30 s

[Note]:

  1. a) Annealing/Extension: The temperature and time can be adjusted according to experimental requirements.
  2. b) Fluorescence signal acquisition: Set the experimental procedure according to the instructions of the instrument. Common instrument settings for different durations are as follows:

20 sec: Applied Biosystems 7700, 7900HT, 7500 Fast

31 sec: Applied Biosystems 7300

32 sec: Applied Biosystems 7500

 

Notes

  1. Ensure the product is completely thawed and thoroughly mixed before use. Briefly centrifuge and collect at the bottom of the tube, then store on ice before use.
  2. To avoid cross-contamination between samples and aerosol contamination, it is recommended to prepare reaction systems in a clean bench.
  3. Avoid repeated freeze-thaw cycles during use.
  4. For your safety and health, wear lab coats and disposable gloves during operation.

Experimental Methods:

Primer Design

  1. Primers must be specific.
  2. The Tm value of primers should be between 58-60°C, and the difference in Tm values between primer pairs should be controlled within 1-2°C.
  3. The length of primers is generally between 15-30 bp.

Probe Design

  1. The Tm value of TaqMan probes should be about 10°C higher than the corresponding primers (68-70°C).
  2. TaqMan probes should not form dimers with primers.
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