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RT² First Strand Kit (12)

Catalog Number Product Description Price  
330401  Reagents for Twelve (12) First Strand cDNA Synthesis Reactions, 20 µl each
Description
The RT² First Strand Kit provides a rapid and convenient procedure for efficient first strand cDNA synthesis. The kit also contains an effective genomic DNA elimination step and a built-in External RNA Control. This all-in-one kit has been designed and optimized for real-time PCR-based gene expression analysis with SABiosciences' RT² Profiler™ PCR Arrays and RT² qPCR Primer Assays. The kit includes a proprietary procedure to effectively eliminate contaminating genomic DNA from RNA samples before reverse transcription. Random hexamers and oligo-dT prime reverse transcription in an unbiased manner, and a reverse transcriptase synthesizes cDNA product with optimal yield and length. A built-in External RNA Control helps monitor reverse transcription efficiency and test for enzyme inhibitors contaminating your RNA samples when used together with RT² RNA QC PCR Array and RT² Profiler™ PCR Array. The magnesium and nucleotide concentrations and other buffer components are the most compatible with RT² SYBR Green qPCR Master Mixes when used in gene expression analysis with RT² Profiler™ PCR Arrays and RT² qPCR Primer Assays.
Materials Included / Packing List
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 temperature.

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           

Protocol

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.

  1. Briefly (10-15 seconds) spin down all reagents.
  2. Prepare the Genomic DNA Elimination Mixture**:
    1. For each RNA sample, combine the following in a sterile PCR tube:
      Total RNA 25.0 ng to 5.0 µg
      GE** (5X gDNA Elimination Buffer) 2.0 µl
      H2O to a final volume of 10.0 µl

      **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 at 1-888-503-3187.
    2. Mix the contents gently with a pipettor followed by brief centrifugation.
    3. Incubate at 42ºC for 5 min.
    4. Chill on ice immediately for at least one minute.
  3. Prepare the RT Cocktail:
  4. RT Cocktail  1 reaction  2 reactions  4 reactions
    BC3 (5X RT Buffer 3)  4µl 8µl 16 µl
    P2 (Primer and External Control Mix) 1µl 2µl 4 µl
    RE3 (RT Enzyme Mix 3) 2µl 4µl 8 µl
    H2O 3µl 6µl 12 µl
    Final Volume  10µl 20µl 40 µl 

  5. First Strand cDNA Synthesis Reaction:
    1. Add 10 µl of RT Cocktail to each 10-µl Genomic DNA Elimination Mixture.
    2. Mix well but gently with a pipettor.
    3. Incubate at 42ºC for exactly 15 min and then immediately stop the reaction by heating at 95ºC for 5 minutes.
    4. Add 91 µl of H2O to each 20-µl of cDNA synthesis reaction. Mix well.
    5. Hold the finished First Strand cDNA Synthesis Reaction on ice until the next step or store overnight at -20ºC.
  6. 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.