M-MLV(H-) Reverse Transcriptase
Obtain higher cDNA yields than with the deletion mutant
Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]), Point Mutant, is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long (> 5 kb) RNA templates. The lack of RNase H activity is beneficial for this application, as RNase H can begin to degrade templates when incubation times are long, as can occur when long cDNAs are synthesized.
Although many investigators are successful in using M-MLV RT (H +) for analytical and some preparative cDNA applications, reverse transcriptases that lack RNase H activity provide another option for preparing long cDNAs and libraries that contain a high percentage of full-length cDNA.
- RNase H Minus – Provides optimal conditions for preparing full-length cDNA from long RNA templates.
- Temperature stability: The thermostability of this point mutant minimizes the problems associated with the secondary structure.
- Increased polymerase activity: M-MLV RT (H–), Point Mutant, provides higher yields of cDNA compared to the deletion mutant (Cat. # M5301).
- Supplied with 5X reaction buffer: 250 mM Tris-HCl (pH 8.3 at 25 ° C), 375 mM KCl, 15 mM MgCl2, 50 mM DTT.
- Wide working range – Greater tolerance to variations in enzyme and substrate concentrations means greater consistency in performance.
- Synthesis of first-strand cDNA.
- Primer extension.
Purified from E. coli expressing the M-MLV pol gene in a plasmid
Performance and quality tests.
SDS-PAGE purity; endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease assays; and yield and length of cDNA product
Definition of unity
One unit of M-MLV RT is the amount of enzyme required to incorporate 1 nmol deoxyribonucleotide into acid precipitable material in 10 min at 37 ° C using poly (A) oligo (dT) 25 as template-primer.
Unit reaction conditions
50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM DTT, 0.5 mM [3H] dTTP, 0.1 mM poly (A), 0.1 mM oligo (dT) 25 mM, 0.1 mg / ml BSA, and enzyme in 50 µL for 10 min at 37 ° C.