Medino®  Fast Cell Direct SYBR Green RT-qPCR Kit

ProductMedino®  Fast Cell Direct SYBR Green RT-qPCR Kit
Catalog No.LM601013
Storage conditionsThis product should be stored at -25~-15℃ for 6 months.

Description

Product description

Medino® Fast CellDirect SYBR Green RT-qPCR Kit is suitable for RNA extraction of various animal cells (such as cell line adherent cells and suspension cells, primary culture cells, various stem cells, iPS cells, etc.), without RNA, can be directly for qPCR expression analysis, short time, simple operation, low error rate, the shortest 1.5 hours can be efficiently from the template preparation to reverse transcription reaction and gene expression analysis. The kit contains the reverse transcription as well as the fluorescence test Agent, can easily perform gene expression analysis.

 

Components

Cat.No.LM601013
Size40T/100T

 

Instructions

  1. Preparation of the cleavage products
  • Place the reagent to melt at room temperature, turn upside down before use, mix gently well, and use after slightly centrifugation to avoid foaming. No mixing reagent, using an oscillator for mixing The lack of reagents on ice will lead to a decrease in reaction performance.
  • Cells were transferred to a centrifuge tube * and cells * * was collected by centrifugation at 5000 rpm for 2min to fully remove the medium. If adherent cells were grown in 96-well plates, then directly The medium was removed
  • When FCD Washing Buffer 150μL was added to each well, cells were washed by suction and centrifuged at 5000 rpm for 2min for FCD Washing Buffer.
  • Add 48 μL FCD Lysis Buffer solution and 2 μL DNasel solution to each well, mix at room temperature and stand for 5min. After incubation, add 2.5 μL FCD Stop Solution * * *, suck and mix for about 5 times to get the lysis product * * * *. When the number of cells exceeds 1105cells, the lysis residue may be positive Often phenomenon.

[Note]:*                The basic requirement for the number of cells in the 50 μ L lysis system is 1104 per well.                     The kit is 110³ -110scells. If the number of cells is large, it can increase appropriately                                          Amount of            FCD Lysis Buffer solution and DNase I solution.

* *             Different centrifugation conditions for different cells, please centrifuge using the centrifugal                               speed suitable for the cells used.

***             50 μL of lysate requires 2.5 μL of FCD Stop Solution. According to the experiment, the                     amount of FCD Stop Solution should be increased when the amount of lysate increases.

**** When the cell lysis product solution for long-term storage, please place in from-25 to-15℃.

 

  1. Reverse transcription
  • After melting at room temperature for 4×Medino®FCD RT Mix, slightly mix on ice and configure the reaction system according to the table below:
ComponentsVolume (μL)Final concentration
4× Medino® FCD RT Mix5
crackateX 
RNase-free  H₂OUp to 20 

 

  • The pipette gently mixed the prepared reaction solution and performed the reverse transcription reaction according to the table procedure
Temp.Time.
55℃15min
85℃5min

 

  1. Reaction program

The reaction solution was prepared using the following proportions (preparation on ice):

 

ComponentsVolume (μL)Final concentration
2×Medino® FCD qPCR SYBR Master Mix10
Forward Primer (10 μM)0.4200 nM
Reverse Primer (10 μM)0.4200 nM
Reverse transcription productsX 
RNase-free  H₂OUp to 20 

 

Quantitative fluorescence PCR by routine amplification procedures

 

Cycle stepTemp.TimeCycles
Predenaturation95℃30sec1
Denaturation95℃10sec35-40
Annealing60℃30sec
Extension60℃30sec
Melt curve phaseInstrument default settings1

 

Notes

  1. Before use, the frozen components are fully melted and gently mixed before use
  2. During the experiment, please try to use pollution-free consumables to avoid pollution.
  3. This product should avoid repeated freezing and thawing, and strong light exposure should be              avoided when preparing
  4. For your safety and health, please wear a lab coat and disposable gloves during operation.
  5. This product is for research use only
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