| Description |
| The ReactionReady™ First Strand cDNA Synthesis Kit
contains all of the reagents needed for the conversion of RNA into first strand cDNA. This kit was
designed and optimized for PCR based gene expression analysis. It includes random hexamers, for
priming reverse transcription in an unbiased manner, and the M-MMLV reverse transcriptase, to
give a maximum yield of cDNA product. The buffer system is optimized for high cDNA yield; and
more importantly, the magnesium concentration is compatible with our RTē PCR Master Mixes in
the subsequent PCR. Remember to order C-01 with our other PCR reagents for optimal performance. |
| 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 on dry ice. For long-term storage, keep entire
box at -20º C
RNase-free H2O
5X Reverse Transcription Buffer (BC)
Random Primers (P) RNase Inhibitor
(RI)
Reverse Transcriptase (RE) |
| Brief Protocol |
- Prepare An Annealing Mixture. For each reaction, combine the following
into a sterile PCR tube:
|
Total RNA |
0.5 to 5.0* |
µg |
|
|
Buffer P |
1.0 |
µl |
|
|
|
|
|
RNase-free H2O to a final volume of |
10.0
|
µl |
|
* The amount of RNA needed depends on your specific
experimental conditions.
Mix the contents gently with a pipettor followed by brief centrifugation.
Place the mixture in a thermal cycler at 70 ºC for 3 min. Cool to 37 ºC and
keep at that temperature for 10 min.
- Prepare the RT Cocktail: This mixture can be prepared while the Annealing
Mixture is incubating at 37 ºC. Warm the RT Cocktail at 37 ºC for 1 min
before proceeding to the next step.
| Number of Reactions: |
1 |
2 |
4 |
8 |
| Buffer BC (5X RT Buffer) |
4
µl |
8
µl |
16
µl |
32
µl |
| RNase-free H2O |
4
µl |
8
µl |
16
µl |
32
µl |
| RI (RNase Inhibitor) |
1
µl |
2
µl |
4
µl |
8
µl |
| RE (Reverse
Transcriptase) |
1
µl |
2
µl |
4
µl |
8
µl |
| Final Volume |
10
µl |
20
µl |
40
µl |
80
µl |
-
First Strand cDNA Synthesis Reaction: Add 10 μl of RT Cocktail to each 10 μl-Annealing Mixture.
Mix well but gently with a pipettor and continue incubation at 37 ºC for 60 min. Heat at 95 ºC for
5 min to hydrolyze the RNA and to inactivate the reverse transcriptase. Hold the finished reaction
on ice until ready to use.
The completed First Strand cDNA Synthesis Reaction can now be used as template for PCR.
|
