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For research purposes only. Not for use in diagnostic procedures for clinical purposes.
Ready-to-use amplification primer mix for RT-PCR using the LightCycler™ Instrument
Human Dex-I (dexamethasone-induced ; MYLE)
Note: After Thawing keep on ice!
material
Vial Label
Content and use
contents
ready-to-use primer mix for target specific Yellow cap
amplification using the LightCycler™FastStart Preparation
achieved with 1µg total RNA isolated with contains optimal MgCl2 concentration andamplification primer pair amplification standard for approximately 34000 Strand cDNA Synthesis Kit (AMV) (Roche).
! The resulting cDNA has to be diluted to a
final volume of 200-500 µl with PCR-
Green cap
grade water
contains a cDNA mix from several humanhematopoietic cell lines Application
Quantitative evaluation of gene expression White cap
Assay time
Set up the PCR amplification 15 min
Additional
1st Strand cDNA Synthesis Kit for RT-PCR (Roche Cat. # 1 483 188) equipment
LightCycler™ FastStart Master SybrGreen I (Roche Cat. # 3 003 230) LightCycler™ PCR run 50 min
LightCycler™ Instrument (Roche Cat. # 2 011 468) LightCycler™ Primer Set Housekeeping genes (Search GmbH) reagents
Number of
required
The LightCycler™-Primer Set is tested using The LightCycler™-Primer Set allows to perform quantitative RT-PCR using the LightCycler™ instrument. An optimized primer pair has been selected for specific amplification oftargets. The amplicon is detected by fluorescence using the Kit storage/
The unopened kit is stable at –20˚C 12 month double-stranded DNA binding dye Sybr®Green I.
stability
Specificity
The LightCycler™-Primer Set “Dex-I” isspecific for the sequence of human Dex-I.
The primers were selected in the first exon of the gene based on their sensitivity, since dueto the presence of a pseudogene onchromosome 16, genomic DNA will bedetected also with intron overspanningprimers. However, no genomic signal will begenerated if RNA or mRNA is generated asdirected (DNAse treatment). If the samplequality is poor or unknown a no-RT controlreaction is strongly recommended.
Amplification
Introduction
A fragment of the human Dex-IcDNA sequence is amplified and Parameter
Additional
reagents
required
Thawing the
solutions
Melting Curve Analysis
LightCycler™ FastStart vial 1a/b
Parameter
It is recommended to define the
experimental protocol before
preparing the solutions
Experimental
Protocol
Program 1: Denaturation ofthe template and activation of Parameter
Denaturation
Parameter
Preparation of Depending on the total number of
Typical results
the master
Introduction
five additional reactions(Standard).
Quantification
in the QC sheet were obtained byperforming the describedprocedure with the enclosed Step Action
standards and control cDNA. Thefluorescence values versus cycle Prepare a fresh dilution series of the standard In a 1.5 ml light protected reaction tube onice, add the following components in theorder mentioned below: Melting curve
Component
LightCycler™ Primer Set (yellow cap) 2 µl
Total Volume
• Pipet 10 µl PCR mix into the precooled
• Add 10 µl of cDNA template
• Pipet 10 µl of PCR mix into 4 precooled
• Add 10 µl of undiluted and of the freshly
Seal each capillary with a stopper and place the adaptors, containing the capillary, into a benchtop microcentrifuge. Centrifuge at 2000 Place capillaries in the rotor of theLightCycler™ Instrument.
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Source: http://www.search-lc.de/lc/info/Dex-I.pdf