The Whole Transcript (WT) Sense Target Labeling Assay Has an Updated Protocol
The GeneChip® WT (Whole Transcript) Terminal Labeling Kit is optimized to be used for the fragmentation and labeling steps of the GeneChip® WT Sense Target (ST) Labeling Assay.
The WT Terminal Labeling Kit employs a strategy for reproducible DNA fragmentation with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1). The typical input material is random-primed, single-stranded DNA, which can be obtained using the GeneChip® WT cDNA Synthesis and Amplification Kit (available separately from Affymetrix) that incorporates deoxyuridine at predefined ratios relative to thymidine.
Following fragmentation, the resulting fragmented DNA can then be labeled using terminal deoxynucleotidyl transferase (TdT) in the presence of a proprietary biotinylated compound, GeneChip® DNA Labeling Reagent. Both components are also provided in the WT Terminal Labeling Kit. The final labeled DNA target is ready to be added to the cocktail for hybridizing to the arrays such as the GeneChip® Human Exon 1.0 ST Array.
|
|
|
10X cDNA
Fragmentation Buffer
|
48 µL |
144 µL |
| UDG, 10 U/µL |
10 µL |
30 µL |
| APE 1, 1,000 U/µL |
10 µL |
30 µL |
| 5X TdT Buffer |
120 µL |
360 µL |
| TdT, 30 U/µL |
20 µL |
60 µL |
| DNA Labeling Reagent, 5 mM |
10 µL |
30 µL |
| RNase-free Water |
825 µL |
2x 825 µL |
The GeneChip® WT Terminal Labeling Kit is also available as a component of the GeneChip® WT Sense Target Labeling and Control Reagents Kit. |
