AMV Reverse Transcriptase: Datasheet
Applications:
- RT PCR
- Synthesis of cDNA
- RNA Sequencing
Description:
AMV Reverse Transcriptase (AMV RT) catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids. The enzyme possesses an intrinsic RNase H activity. AMV RT possesses multiple enzymatic activities including RNA- and DNA-directed DNA polymerase, DNA-RNA unwinding activity, a sequence-specific Mn2+-dependent endonuclease and ribonuclease H.
Concentration: 10 u/µl
Storage Buffer:
200mM potassium phosphate (pH7.2), 0.2% Triton X-100, 2mM DTT and 50% glycerol
Reaction Buffer 5X:
250mM Tris-HCl (pH8.3 @ 250 C), 250mM KCl, 50mM MgCl2, 2.5mM spermidine and 50mM DTT
Unit definition:
One unit is the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37°C. Reaction conditions are: 50mM Tris-HCl (pH 8.3), 8.75 mM MgCl2, 40 mM KCl, 10 mM DTT, 0.1 mg/ml BSA, 1 mM radiolabelled dTTP and 0.25mM poly(A):oligo(dT).
Quality control:
First-Stand cDNA Synthesis: First strand cDNAs, of 1.2 kb Control RNA is synthesized using 30 units of enzyme, 1 µg of each template, an oligo(dT) primer and a radiolabelled dNTP. The minimum specification is the production of 120 ng of first-strand cDNA. Full-length cDNA must be observed by gel electrophoresis and autoradiography.
Endonuclease Activity: 1 µg of Type I supercoiled plasmid DNA is incubated with 25 units of enzyme in 50mM Tris (pH8.3), 40mM KCl, 7mM MgCl2, 10mM DTT for one hour at 370C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.
Nuclease Activity: 50 ng of radiolabelled DNA or RNA is incubated with 25 units of enzyme in 4 mM Tris (pH8.3), 3.2 mM KCl, 0.56 mM MgCl2, 0.8 mM DTT for one hour at 37°C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Passing specifications is <1% release for DNase and <3% release for RNase.
Usage:
Standard Protocol:
We recommend to prepare 2 Mixes
Mix I
|
Component |
Amount/conc. |
RNA
or
polyA RNA |
2 µg
50-500 ng |
|
primer |
500 ng for each µg of RNA |
sterile Water
gently vortex |
up to 8 µl (max 11µl)
|
|
Incubation |
Temperature |
|
5 min |
70 °C |
|
5 min |
chill on ice |
Mix II
|
Component |
Amount/conc. |
|
AMV 5X reaction buffer |
5 µl |
|
dNTP mix (10 mM of each = 40 mM) |
2.5 µl |
|
optional: RNAsin |
20-40 units |
|
sodium pyrophosphate (40 mM) @ 42°C |
2,5 µl ( |
|
AMV Reverase (10 u/µl) |
3 µl (30 units) |
|
sterile water |
up to 25 µl |
|
combine Mix I and Mix II and vortex gently |
|
prepare a tube containing containing fresh 2–5 μCi [α-32P]dCTP
Transfer 5 µl of the master mix (Mix I and Mix II) to that tube (Mix III) |
|
Incubate
60 min for Oligo(dT) primers
or
60 min for random hexamer primer |
42°C
37°C |
|
after incubation: place the samples on ice |
|
Add 95 µl of 50 mM EDTA to Mix III
(can be used for gel analysis) |
|
Perform second-strand synthesis using
the unlabeled first-strand reaction |
|
Transportation: on blue ice
Storage: at -20°C for 24 months
|
