The GeneChip® WT (Whole Transcript) cDNA Synthesis and Amplification Kit is optimized to be used in the first steps of the GeneChip® WT Sense Target (ST) Labeling Assay.
The WT cDNA Synthesis and Amplification Kit supports the first-and second-cycle cDNA synthesis as well as the intermediate in vitro transcription amplification procedures.
The input material is 1 µg of total RNA (1 µg recommended, with an acceptible range of 1-2 µg), which is treated with the RiboMinus™ Human/Mouse Transcriptome Isolation kit (from Invitrogen, not included in this kit) to remove the majority of the ribosomal RNA for enhanced sensitivity. The resulting product from the WT cDNA Synthesis and Amplification Kit is random-primed, single-stranded DNA in the sense orientation, ready to be fragmented and labeled with the GeneChip® WT Terminal Labeling Kit (available separately from Affymetrix).
An optional method may be used to reduce the starting material to 100 ng of total RNA (100 ng recommended, with an acceptible range of 100-300ng).
Sub-Kit 1: GeneChip® WT cDNA Synthesis Kit
For 10 Rxns
For 30 Rxns
T7-(N)6 Primers, 2.5 µg/µL
20 µL
60 µL
5X 1st Strand Buffer
100 µL
300 µL
DTT, 0.1M
50 µL
150 µL
dNTP, 10 mM
9µL
27µL
RNase Inhibitor
25µL
75µL
SuperScript™ II
90 µL
270µL
MgCl2, 1M
40 µL
120 µL
DNA Polymerase I
6µL
18µL
RNase H
22µL
66µL
Random Primers, 3 µg/µL
20 µL
60µL
dNTP+dUTP, 10 mM
20 µL
60 µL
RNase-free Water
4.44 mL
4.44 mL
Sub-Kit 2: GeneChip® WT cDNA Amplification Kit
For 10 Rxns
For 30 Rxns
10X IVT Buffer
50 µL
150 µL
IVT NTP Mix
200 µL
600 µL
IVT Enzyme Mix
50 µL
150 µL
IVT Control
10µL
10µL
The WT cDNA Synthesis and Amplification Kit is also available as a component of the GeneChip® WT Sense Target Labeling and Control Reagents Kit.
ORDERING INFORMATION
Assays and Reagents
Part #
Description
900673
GeneChip® WT cDNA Synthesis and Amplification Kit
Sufficient for 30 reactions
900652
GeneChip® WT Sense Target Labeling and Control Reagents
