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)
After Thawing keep on ice!
Content and use
ready-to-use primer mix for target specific
amplification using the LightCycler™FastStart
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-
contains a cDNA mix from several humanhematopoietic cell lines
Quantitative evaluation of gene expression
Set up the PCR amplification 15 min
1st Strand cDNA Synthesis Kit for RT-PCR (Roche Cat. # 1 483 188)
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)
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
The unopened kit is stable at –20˚C 12 month
double-stranded DNA binding dye Sybr®Green I.
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.
A fragment of the human Dex-IcDNA sequence is amplified and
Melting Curve Analysis
LightCycler™ FastStart vial 1a/b
It is recommended to define the
experimental protocol before
preparing the solutions
Program 1: Denaturation ofthe template and activation of
Depending on the total number of
five additional reactions(Standard).
in the QC sheet were obtained byperforming the describedprocedure with the enclosed
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:
LightCycler™ Primer Set (yellow cap) 2
• 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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