| Description
Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus, Point Mutant (M-MLV RT ( H- )) is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long RNA templates (>5kb) . The lack of RNase H activity is beneficial for this application, as RNase H can start to degrade templates when incubation times are long, as they may be when making long cDNAs. Although many researchers are successful in using M-MLV RT(H+) for analytical and some preparative cDNA applications, reverse transcriptases lacking RNase H activity provide another option for the preparation of long cDNAs and for libraries containing a high percentage of full-length cDNA.
Quality Control
1. Nickase: Greater than 90% supercoiled plasmid remains after incubation with 200 units of enzyme for 60 minutes at 37 ¡æ .
2. DNase Assay: 200 units of enzyme are incubated with 50ng of 3 H-DNA for 60 minutes at 37 ¡æ . Minimum passing specification is less than 1% release.
3. RNase Assay: 200 units of enzyme are incubated with 50ng of 3 H-RNA for 60 minutes at 37 ¡æ . Minimum passing specification is less than 3% release.
4. First-stand cDNA reaction: Using 200 units of enzyme in a standard reaction, isotope is incorporated into cDNA from 1¦Ì g of a 1.2 kb RNA and 1¦Ì g of a 7.5 kb RNA. By autoradiography, the cDNA product is a single, full-length band for the 1.2 kb RNA and 25% full length product for the 7.5 kb RNA. The minimum passing specification is 12% conversion of both RNAs into cDNA.
5. Rnase H Activity: 200 units of enzyme are incubated with poly A: polydT for 60 minutes at 37 ¡æ . Minimum passing specification is less than 1% release.
6. Physical Purity: >90% pure on SDS PAGE. Features
1. RNase H Minus: Provides optimal conditions for the preparation of full-length cDNA from long RNA templates.
2. Temperature Stability: Thermostability of this point mutant prevents problems associated with secondary structure.
3. Increased Polymerase Activity: M-MLV RT (H-), Point Mutant, gives higher yields of cDNA compared with the deletion mutant.
4. Provided with 5¡Á Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25 ¡æ ), 375mM KCl, 15mM MgCl 2 , 50mM DTT.
5. Broad Working Range : More tolerance to variations in enzyme and substrate concentration means improved consistency in performance.
References
Roth, M.J. et al. (1985) J. Biol. Chem. 260 , 9326-35.
Applications
First strand cDNA synthesis.
Primer extension. Order Information
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