QuantiSure Flex cDNA Synthesis Kit
Cat. No. |
Size |
Price |
Quantity |
11021-25 |
25 x 20-µl reactions |
$119.00 |
|
11021-50 |
50 x 20-µl reactions |
$199.00 |
|
11021-200 |
200 x 20-µl reactions |
$769.00 |
Benefits
- Flexibility in priming methods: oligo(dT), random primer, or gene-specific primer.
- Highly efficient reverse transcription from different amounts of RNA template.
- Robust and reproducible reactions.
- Easy reaction setup.
Description
QuantiSure Flex cDNA Synthesis Kit is a highly efficient and complete system for the synthesis of first strand cDNA. This kit provides flexible procedure for reverse transcription using different priming strategies including oligo(dT), random primer, or gene-specific primer. Two key components in this kit are RT Enzyme Mix and Reaction Buffer. The RT Enzyme Mix is a mixture of MMLV reverse transcriptase, and recombinant RNase inhibitor, which effectively protects RNA template from degradation, while Reaction Buffer contains optimized buffer, magnesium, dNTPs, and stabilizers. This kit also contains Random Primer and Oligo(dT) to meet your specific needs for reverse transcription. In general, Oligo(dT) and gene-specific primer can be used to produce cDNA more than 10 kb. Random primer yields shorter cDNA on average (~1 kb) and can be used for the detection of multiple short RT-PCR products. This easy-to-use kit simplifies reaction set-up and ensures reproducible synthesis of first strand cDNA from total RNA or polyA+ RNA template.
Components
Component |
Description |
Reaction Buffer (5X) |
Buffer optimized for reverse transcription; Contains MgCl2, dNTPs, and stabilizers |
RT Enzyme Mix |
Mixture of reverse transcriptase, and RNase inhibitor. |
Random Primer |
10x concentrated solution of random primer |
Oligo(dT) |
10x concentrated solution of Oligo(dT)18 |
RNase-free H2O |
Ultrapure quality |
Storage
The kit is stable for one year when stored in a constant temperature freezer at -20ºC. After thawing, mix the components thoroughly before using. Frequent freezing and thawing is not recommended.
Quick-Reference Protocol
1. Thaw all components. Mix each solution, centrifuge briefly, and then store on ice.
2. Mix template RNA and primer solution in a nuclease-free tube on ice.
Component |
Volume/Reaction |
Template RNA |
Variable (up to 1 µg total RNA) |
Oligo(dT) or Random Primer * |
2 µl |
RNase-free H2O |
Variable |
Total Volume |
14 µl |
* Combination of 2 µl Oligo(dT) and 2 µl Random Primer can be used for a mixed priming method. If gene-specific primers are used, a final concentration of 0.7 µM is recommended.
3. Incubate for 5 min at 65ºC, then place immediately on ice.
4. Briefly centrifuge the tube from step 3, and place on ice. Add the following components for a total reaction volume of 20 µl.
Component |
Volume/Reaction |
Reaction Buffer (5 X) |
4 µl |
RT Enzyme Mix |
2 µl |
Total Volume |
20 µl |
5. If Oligo(dT) or gene-specific primer are used, incubate for 1 hour at 42ºC.
If Random Primer is used, incubate for 5 min at 25ºC followed by 1 hour at 42ºC.
6. Incubate for 5 min at 80°C to stop the reaction.
7. Place finished reverse-transcription reaction on ice until ready to use, or at -20ºC for long-term storage.
Note: The resulting cDNA can be used for real-time PCR or end-point PCR. The volume of the cDNA added (from the undiluted RT reaction) should not exceed 1/10 of the final PCR volume.