M-MLV Reverse Transcriptase is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. Compared to AMV RT, Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) lacks DNA endonuclease activity and has a lower RNase H activity.
cat. no.
amount
note
STS-MRT 10
10000 units
5X Reverse Buffer
STS-MRT 50
50000 units
5X Reverse Buffer
STS-MRT 200
200000 units
5X Reverse Buffer
FOR RESEARCH USE ONLY
SHIPPING
Shipped in green ice.
FORM
Liquid
SHELF LIFE
12 months
UNIT DEFINITION
One unit is defined as the amount of enzyme required to incorporate 10 nanomoles of dNTPs into acid-insolubile material in 30 min at 74°C.
CONCENTRATION
250 units/μl
STORAGE
Store at -20°C
Components
component
STS-MRT 10
STS-MRT 50
STS-MRT 200
M-MLV Reverse Transcriptase
5X Reverse Buffer
10000 units / 50ul
1 ml 5X Buffer
10000 units / 250ul
1 ml 5X Buffer
200000 units / 1 ml
5 ml 5X Buffer
DTT
500 ul / 100mM
500 ul / 100mM
500 ul / 100mM
Assay set up
First Strand cDNA synthesis
Assay Set-Up:
Before starting, vortex all components thoroughly to ensure homogeneity.
Prepare a premix for the number of assays you need according to the following protocol:
First - Strand cDNA synthesis
Final volume: 20ul
- In a nuclease-free microcentrifuge tube add:
- dNTPs mix (10mM each) 1ul
- 1ng to 4ug total RNA
or
1-500ng mRNA xul
- 50-100ng random primers
or
Oligo(dT)12-18 (500ug/ml) 1ul
- ddH2Oup to 12ul
- Heat mixture @ 65°C for 2min. and quick cool the tube on ice. Add:
- 5X Reverse Buffer 4ul
- 100mM DTT 2ul
- ddH2O 1ul
or
Ribonuclease Inhibitor
(optional)
- Mix gently and incubate
- @ 42°C for 2 min. if you are using oligo(dT)12-18
or
- @ 25°C for 2 min. if you are using random primers
- Add 1ul (200U) of GeneSpin M-MLV Reverse Transcriptase
(if you are using random primer incubate @25°C for 10 min.)
- Incubate @ 42°C for 60 - 120 min.
- Inactivate the reaction @ 70°C for 10 min.
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