EB0118 M-MLV Reverse Transcriptase

M-MLV Reverse Transcriptase 4.0

Cat#EB0118

Description

Reverse transcriptase 4.0 is a fourth-generation M-MLV reverse transcriptase obtained through gene modification and recombination techniques. Compared with the previous M-MLV reverse transcriptase, this enzyme has improved its tolerance to inhibitors that interfere with cDNA synthesis, and can perform stable specific cDNA formation on samples from different sources. At the same time, the reaction is faster, the incubation time has been reduced from more than 50 minutes to 10 minutes, and the ability to continuously synthesize (end product 12.3 kb) has been improved over the previous version.

Product Composition

Storage Conditions

Store at -80 ℃. It can be stored at this temperature for one year. Avoid exposure to frequent temperature changes.

Usage

The enzyme can be used for fluorescent RT-RPA or RPA, depending on the optimization degree of primer screening and detection means, the detection limit is 10-100 copies/test, and the reaction time is not more than 15 minutes.

Stability

When placed at -80 ℃ for 3 months, the enzyme application activity did not decrease significantly (data update).

Freezing and thawing for 5 times showed no significant decrease in enzyme activity.

The enzyme application activity did not decrease significantly after being placed at 4 ℃ for 21 days (data update).

After lyophilization, the enzyme application activity did not decrease significantly.

Quality Control

Protein Purity

The purity of reverse transcriptase 4.0 was detected by molecular exclusion high performance liquid phase.

Endonuclease Activity

The 50 μL reaction system consisted of 10 μL reverse transcriptase 4.0 and 500 ng circular plasmid DNA. After incubation at 37 ℃ for 4 h, the endonuclide activity was detected by agar-gel electrophoresis.

Exonuclease activity

The 50 μL reaction system consisted of 10 μL reverse transcriptase 4.0 and 500 ng double-stranded DNA fragments, and was incubated at 37 ℃ for 4 hours. The exonuclide activity was detected by agar-gel electrophoresis.

RNA hydrolase activity

The 50 μL reaction system consisted of 10 μL reverse transcriptase 4.0 and 500 ng single-stranded RNA, and was incubated at 37 ℃ for 4 hours. The RNA hydrolase activity was detected by agarose gel electrophoresis.

Host Residual DNA

Escherichia coli 16S rDNA was used as the target to detect the residual amount of host DNA in recombinase by fluorescence quantitative PCR.

Application Example

Enzyme Activity Assays

Figure 1. Amplification sensitivity of different types of templates. The RT-RPA reaction system consisted of recombinase, recombinase helper protein, single-stranded DNA binding protein, DNA polymerase, reverse transcriptase 4.0, exonuclease III, RT-PRA buffer, PEG and trehalose. The reaction was performed in a qPCR apparatus at 42 ℃ for 25 minutes. (A) A RNA virus template; (B) A DNA plasmid template.

Enzyme stability test

Figure 2. Stability of reverse transcriptase. Reverse transcriptase was freeze-dried in storage solution (A) and placed at 4 ℃ for 21 days (B). The enzyme stability was determined by RT-RPA reaction system. Template: RNA virus, template size: 10⁴ and 10³ copies/reactions.