lyophilised cDNA Master Mix
For in vitro use only!
Shipping: shipped at ambient temperature
Storage Conditions: store at ambient temperature
Store in an aluminium-coated bag or on a dry place.
Lyophilisates may hydrate at humidity levels >70 % when sealing is opened.
Shelf Life: 12 months
RT Master Lyophilisate contains a genetically engineered version of M-MLV Reverse Transcriptase (M-MLV RT) with eliminated RNase H activity and increased thermal stability. The enzyme is a RNA-directed DNA polymerase that synthesizes a complementary DNA strand initiating from a primer using single-stranded RNA or DNA as template. Its enhanced thermal stability in combination with the deactivated RNase H activity results in an increased specificity, higher cDNA yield and an improved efficiency for full length cDNA synthesis compared with standard M-MLV RT. The enzyme is recommended for synthesis of cDNA from 100 bp up to 10 kb length.
RT Master Lyophilisate
Preloaded lyophilisates containing SCRIPT Reverse Transcriptase, dNTPs, Reaction Buffer, MgCl2 and stabilizers.
PCR grade water
RT Master Lyophilisate is delivered in reaction tube strips or 96-well plates preloaded with a complete RT master mix including dNTPs in a dry, room temperature stable format. The lyophilisate combines highest performance with convenience of use and stability. There is no need for freezing, thawing or pipetting on ice. The few remaining pipetting steps minimize the risk of errors or contaminations.
Each vial contains all components (except primers and template) required for a 20 μl reverse transcription assay. To perform the assay, only fill up the vials with a mix of primers and RNA template.
Recommended protocol for cDNA synthesis
1. Preparation of the RNA/Primer Mix
Add the following components to a nuclease-free microtube and mix by pipetting gently up and down:
|RNA template||10 pg - 5 μg total RNA or 10 pg - 500 ng mRNA|
|Primer1)||gene-specific primer: 10-20 ng (2-4 pmol)|
oligo-dT15-25 primer: 200-500 ng (30-75 pmol)
random primer: 50-200 ng (30-120 pmol)
|RNase-free water||fill up to 20 μl|
3. Dispensing the master mix
Dispense 20 μl of the RNA/Primer Mix to each lyophilisate containing tube or well of the plate.
Incubate the reaction mix at 50°C for 30-60 min.
Please note: The optimal time depends on the length of cDNA. Incubation of 60 min is recommended for cDNA fragments of more than 2,000 bp length. The optimal temperature depends on the structural features of the RNA. Increase the temperature to 55°C for difficult templates with high secondary structure. Note that optimal reaction time and temperature should be adjusted for each particular RNA.
Heat the mixture to 75°C for 15 min to inactivate the Reverse Transcriptase.
6. RNA removal (if required)
The cDNA can now be used as template for amplification in PCR. However, some specific DNA amplifications may requires the prior removal of RNA. Add 2 units RNase H and incubate at 37°C for 20 min.