Thermostable MMLV Reverse Transcriptase (RNase H-)

Descripción del producto

Product Description:

Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) can catalyze the polymerization of cDNA using template single strand DNA or RNA.

This Thermostable MMLV reverse transcriptase(RNase) is a mutant of MMLV reverse transcriptase. It removes the active center of RNase H through point mutations so that to reduce the activity of RNase H, improve the thermal stability of enzyme,  and enhance the  affinity of template, thus reduce the degradation of RNA in reverse transcriptase reaction and increase the yield of first strand cDNA , so it is easier to obtain fulllenght cDNA. At the same time improves the thermal stability, the optimal reaction temperature *0 °c.

Product concentration: **0u/μl

Storage conditions: store at *0°c, shelf life more than a year. Avoid exposure to frequent temperature changes.

Source: Purified from an E. coli strain expressing a recombinant clone, wiht Moloney murine leukemia virus (MMLV) reverse transcriptase gene.

Product Purity: to incubate **0U of this enzyme and 1 μg rRNA (*6s,*3s) for 1 hour at *7°c,  the electrophoresis bands with no visible change.

[5×RT Buffer provided]

**0 mM TrisHCl(pH8.3),

*5 mM MgCI2,

**5 mM KCI,

*0mM DTT

Standard applications:

A. Firststrand synthesis of cDNA

B. RT PCR reaction

Reaction Conditions Recommended:

A. Firststrand synthesis of cDNA:

1.In a RNasefree PCR tube, add 1μl of Oligo(dT)***8(1ug/ul) or *0ng5ug of random primers (****0ng) ,xul of total RNA(*0ng5μg) or mRNA (***0ng), 1ul of dNTP(*0mM each), (*3x ul) of DEPC ddOH2 and heat the tube to *0°c for *0 minutes to melt secondary structure within the template. Cool the tube immediately on ice for 2~*0 minutes to prevent secondary structure from reforming, then spin briefly to collect the solution at the bottom of the tube.

2. Add 4ul of 5×RT Buffer, 1ul of RNasin (*0U/ul) (optional), mix gently by flicking the tube and heat the tube to *2°c for 2 minutes for Oligo (dT)***8 and primeradaptors (or *0 minutes at *5°c for random primers)

3. Add **0U RTase , and incubate for *0 minutes at *2°c.

4. Heat the tube to *0°c for *5 minutes. Then cool the tube immediately on ice to obtain cDNA. The product can be directly used for the second strand synthesis of cDNA or RT PCR amplification reaction, please store at *0°c.

Amplification effect chart:

In the above chart, template is the mouse total RNA , the reverse transcription temperature *0 °c, A1,  A2 is this enzyme (**0U, Part No.JLE****3), B1, B2 and C1, C2 are similar products from other famous company.