Medino®  Fast Cell Direct Probe RT-qPCR Kit

ProductMedino®  Fast Cell Direct Probe RT-qPCR Kit
Catalog No.LM602007
Storage conditionsThis product should be stored at -25~-15℃ for 1year.

Description

Product description

Medino® Fast Cell Direct Probe RT-qPCR Kit is suitable for quantitative gene expression analysis of various animal cells (such as cell line adherent cells and suspended cells, primary culture cells, various stem cells, iPS cells), without RNA, can be directly for qPCR expression analysis, short time, simple operation, low error rate, the shortest only 1.5h can be efficiently from template preparation to transactivation reaction and gene expression analysis steps. The kit contains reverse transcription as well as a quantitative test Agent, can easily perform gene expression analysis.

 

Components

Cat.No.LM602007
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. Cells can be aspirated directly if cultured in 96-well plates culture medium..
  • 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 μLFCD Stop Solution * * * 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 50 μL lysis system is 110² -1105 cells                                        per well. If the number of cells is large, FCD Lysis Buffer solution and DNase I can be                                      increased in equal proportion The amount of 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 FCDStop Solution should be increased when the amount of lysate                      increases.
**** When the cell lysis product solution is stored for a long time, please place it in-25~15℃.

 

  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.
37℃5min
85℃5sec

 

  1. Reaction program
  • The reaction solution was prepared using the following proportions (preparation on ice):
ComponentsVolume (μL)Final concentration
2× Medino® FCD Probe qPCR Mix12.5
Forward Primer (10 μM)0.5200 nM
Reverse Primer (10 μM)0.5200 nM
Probe (10 μM)0.25-1100-400 nM
Reverse transcription productsX 
RNase-free  H₂OUp to 25 

 

  • This product recommends the use of rapid program amplification, which can greatly reduce the reaction time. The annealing temperature can be adjusted when it is difficult to amplify
Cycle stepTemp.TimeCycles
Predenaturation95℃10sec1
Denaturation95℃5sec45
Annealing60℃10sec
Extension60℃10sec

 

  • Regular procedures can also be used for amplification when the experimental instrument does not meet the rapid reaction time.
Cycle stepTemp.TimeCycles
Predenaturation95℃10sec1
Denaturation95℃15sec40-45
Annealing60℃30sec
Extension60℃30sec

 

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.

This product is for research use only

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