AMV RT:
The optimal incubation temperature of a cDNA reaction using AMV RT is 42ºC. Use of the standard 10 X Reaction Buffer results in a final concentration of 5 mM MgCl2at 1X. At this concentration of MgCl2, the endogenous RNaseH of AMV RT is minimal.
AMV RT is naturally more thermostable than other RT’s because of its origin from a chicken virus, whose host’s body temperature is 42ºC. However, it is usable at temperatures of up 58ºC, using modified protocol (above).
Recommended doses of AMV RT into cDNA reactions depends on the application, and the amount of primed RNA to be transcribed.
3.1 For one µg of polyadenlyated RNA, use 5 units of AMV RT in a 25 uL reaction.
3.2. For making a cDNA library, use 5 units of AMV RT per µg of RNA in a 25 uL reaction.
3.3. For RT-PCR, use the same guidelines in 1 and 2, then heat inactivate the RT for 5 minutes at 90ºC, then use an aliquot of the reaction for PCR.
Potency of LSAT AMV RT
It should be noted by the user that AMV Reverse Transcriptase from LSAT is very robust in the potency and purity of the product. It has been repeatedly documented that when compared in quantitative kinetic assays with AMV RT’s from well-known scientific houses, that the LSAT unit is at least five times more potent than that of others. This results in higher yields of cDNA and greater longevity of LSAT enzyme in your freezer.
Quality Control Testing:
All lots of AMV RT are stringently tested for exogenous Ribonuclease and Deoxyribonuclease, and are virtually nuclease free. For testing details and specifications, see below:
Deoxyribonuclease Activity:
50 units of AMV RT XL (or 30 units of AMV RT) are mixed with 0.5 µg of Hae fragments of Phi X174 DNA and incubated for 3 hours at 37ºC in a reaction buffer containing: 10 mM TrisHCl, pH 7.5, 5 mM MgCl2,and 1 mM CalCl2. No more than an equivalent of 1.25 x 10 E-4 unit of DNase 1 is detected.
Ribonuclease Activity:
Twenty units of AMV RT XL (or fifteen units of AMV RT) was incubated with an one microgram of RNA ladder (0.5 – 9.0 Kb) in 1X ARB 45 buffer. Electrophoretic analysis of the RNA in an agarose gel indicated no greater than the equivalent of 8 X 10-8 units of RNase 1A. This assay is capable of detecting 2 X 10-8 unit of RNAse 1A.
cDNA synthesis:
In a 25 uL reaction, one microgram of a poly-adenylated RNA ladder is primed with 0.5 ug of p(dT)12-18, then incubated with 1.0 mM dNTP’s in 1X ARB reaction buffer (Cat. # ARB 45) and 5 units of AMV RT XL at 42ºC for one hour. [32-P]-dATP is included in the reaction. The percent transcription is evaluated by TCA-precipitation, and an aliquot is alkalized and electrophoresed in an alkaline agarose gel.
The gel is then dried and exposed to X-Ray film to generate an electrophoregram. The dried gel is fractionated and the bands of cDNA evaluated by Cerenkov counting. The lot-specific data are stated of the Certificate of Analysis of each lot of RT-XL. At least 60-100% of the poly-A RNA is transcribed, with cDNA.
References:
Hellman, G.M., Shahabuddin, M., Shaw, J.G.., and Rhoads, R.E. (1983) Virology 128:210-220
Houts, G. E.., Miyagi, M., Ellis, C., Beard, D., and Beard, J.W. (1979) J. Virol. 29:517-522
McDonnell, M.S., Simon, M.N., and Studier, F.W. (1977) J. Mol. Bio. 110:119-146
Locker, J. (1979) Anal. Biochem. 98:358-367
Shimomaye, E., and Salvato, M. (1989) Gene. Anal. Tech. 6
AMV RT:
- The optimal incubation temperature of a cDNA reaction using AMV RT is 42ºC. Use of the standard 10 X Reaction Buffer results in a final concentration of 5 mM MgCl2at 1X. At this concentration of MgCl2, the endogenous RNaseH of AMV RT is minimal.
- AMV RT is naturally more thermostable than other RT’s because of its origin from a chicken virus, whose host’s body temperature is 42ºC. However, it is usable at temperatures of up 58ºC, using modified protocol (above).
- Recommended doses of AMV RT into cDNA reactions depends on the application, and the amount of primed RNA to be transcribed.
3.1 For one µg of polyadenlyated RNA, use 5 units of AMV RT in a 25 uL reaction.
3.2. For making a cDNA library, use 5 units of AMV RT per µg of RNA in a 25 uL reaction.
3.3. For RT-PCR, use the same guidelines in 1 and 2, then heat inactivate the RT for 5 minutes at 90ºC, then use an aliquot of the reaction for PCR.
Potency of LSAT AMV RT
It should be noted by the user that AMV Reverse Transcriptase from LSAT is very robust in the potency and purity of the product. It has been repeatedly documented that when compared in quantitative kinetic assays with AMV RT’s from well-known scientific houses, that the LSAT unit is at least five times more potent than that of others. This results in higher yields of cDNA and greater longevity of LSAT enzyme in your freezer.
Quality Control Testing:
All lots of AMV RT are stringently tested for exogenous Ribonuclease and Deoxyribonuclease, and are virtually nuclease free. For testing details and specifications, see below:
Deoxyribonuclease Activity:
50 units of AMV RT XL (or 30 units of AMV RT) are mixed with 0.5 µg of Hae fragments of Phi X174 DNA and incubated for 3 hours at 37ºC in a reaction buffer containing: 10 mM TrisHCl, pH 7.5, 5 mM MgCl2,and 1 mM CalCl2. No more than an equivalent of 1.25 x 10 E-4 unit of DNase 1 is detected.
Ribonuclease Activity:
Twenty units of AMV RT XL (or fifteen units of AMV RT) was incubated with an one microgram of RNA ladder (0.5 – 9.0 Kb) in 1X ARB 45 buffer. Electrophoretic analysis of the RNA in an agarose gel indicated no greater than the equivalent of 8 X 10-8 units of RNase 1A. This assay is capable of detecting 2 X 10-8 unit of RNAse 1A.
cDNA synthesis:
In a 25 uL reaction, one microgram of a poly-adenylated RNA ladder is primed with 0.5 ug of p(dT)12-18, then incubated with 1.0 mM dNTP’s in 1X ARB reaction buffer (Cat. # ARB 45) and 5 units of AMV RT XL at 42ºC for one hour. [32-P]-dATP is included in the reaction. The percent transcription is evaluated by TCA-precipitation, and an aliquot is alkalized and electrophoresed in an alkaline agarose gel.
The gel is then dried and exposed to X-Ray film to generate an electrophoregram. The dried gel is fractionated and the bands of cDNA evaluated by Cerenkov counting. The lot-specific data are stated of the Certificate of Analysis of each lot of RT-XL. At least 60-100% of the poly-A RNA is transcribed, with cDNA.
References:
- Hellman, G.M., Shahabuddin, M., Shaw, J.G.., and Rhoads, R.E. (1983) Virology 128:210-220
- Houts, G. E.., Miyagi, M., Ellis, C., Beard, D., and Beard, J.W. (1979) J. Virol. 29:517-522
- McDonnell, M.S., Simon, M.N., and Studier, F.W. (1977) J. Mol. Bio. 110:119-146
- Locker, J. (1979) Anal. Biochem. 98:358-367
- Shimomaye, E., and Salvato, M. (1989) Gene. Anal. Tech. 6
