Please check the kit components immediately after you receive this
package. SABiosciences is not responsible for missing items not reported within
two (2) business days upon receipt.
Storage Conditions: The following kit is shipped dry ice or blue ice. For long-term storage, keep entire
box at -20º C
Shelf Life: All reagents are
stable for 6 months after receipt of the kit if stored at the recommended
GE: 5X gDNA Elimination Buffer
BC3: 5X Reverse Transcription Buffer 3
H2O: RNase-free H2O
P2: Primer and External Control Mix
RE3: RT Enzyme Mix 3
NOTE: Do not use DEPC treated H2O. Use high-quality,
nuclease-free H2O. If you are not sure whether your RNase, DNase-free
water has been DEPC treated, please check with the supplier.
- Briefly (10-15 seconds) spin down all reagents.
- Prepare the Genomic DNA Elimination Mixture**:
For each RNA sample, combine the following in a sterile PCR tube:
||25.0 ng to 5.0
|GE** (5X gDNA Elimination Buffer)
|H2O to a final volume of
**The RT² First Stand Kit (330401) is not compatible with the chemicals in
Ambion's DNA-free™ kits. If your RNA sample has been treated with
Ambion's DNA-free™ reagents, please call SABiosciences Technical Support
Mix the contents gently with a pipettor followed by brief centrifugation.
Incubate at 42ºC for 5 min.
Chill on ice immediately for at least one minute.
- Prepare the RT Cocktail:
| BC3 (5X RT
| P2 (Primer and External Control
| RE3 (RT Enzyme
First Strand cDNA Synthesis Reaction:
Add 10 µl of RT Cocktail to each 10-µl Genomic DNA
Mix well but gently with a pipettor.
Incubate at 42ºC for exactly 15 min and then immediately stop
the reaction by heating at 95ºC for 5 minutes.
Add 91 µl of H2O to each 20-µl of cDNA
synthesis reaction. Mix well.
Hold the finished First Strand cDNA Synthesis Reaction on ice until
the next step or store overnight at -20ºC.
RNA Quality Control Check (Optional):
If desired, proceed to characterize a small aliquot (6 µl) of the
diluted cDNA template on the correct species-specific and instrument-specific
RT² RNA QC PCR Array following the instructions provided in its User Manual.
Save the remainder at - 20ºC.