HotScript M-MuLV RTase undergoes multiple mutations in M-MuLV, improving its temperature tolerance for reverse transcription. It can be reverse transcribed at temperatures as high as 55 ℃ to 60 ℃ (with activity partially reduced at higher temperature reversals), greatly improving the reverse transcription efficiency of RNA templates with high GC content and rich secondary structures.
HotScript M-MuLV RTase lacks RNase H activity and has stronger elongation and stability compared to wild-type M-MuLV. It can be used for longer cDNA synthesis and the construction of a high proportion of full-length cDNA libraries. The length of synthesized cDNA fragments can reach up to 12kb.
Component 10KU 5 × 10KU
HotScript M-MuLV RTase (200U/ μ L) 50 μ L 5 × fifty μ L
five × RT Buffer 500 μ L 5 × five hundred μ L
Storage: -20 ℃
Avoid RNase contamination.
To ensure successful reverse transcription, it is recommended to use high-quality RNA samples.
If the RNA template has a rich GC content or complex secondary structure, it can be first mixed with RNA template, primer, and RNase free H2O, denatured at 65 ℃ for 5 minutes, cooled on ice, centrifuged briefly, and then added with other components to continue the reverse transcription step below.
The HotScript M-MuLV RTase solution is very viscous and can easily adsorb on the pipe wall and outside the suction head, resulting in loss. Before use, please use it after centrifugation and avoid loss of adhesion on the outer wall of the suction head. Can be adjusted to 0.8 at a time μ The use of L does not affect the effectiveness of use.
1、 First strand cDNA synthesis
Configure reaction system (20 μ L)
Ingredient dosage
Total RNA/mRNA 50ng~5 μ G/5-500ng
Oligo (dT) 18 (0.5 μ G/ μ L)
Or Random Primer (0.1 μ G/ μ L)
Or GSP (Gene Specific Primer) 1 μ L
one μ L
2pmol
DNTP Mixture (10mM each) 1 μ L
five × RT Buffer 4 μ L
RNase Inhibitor (40U/ μ L) 0.5 μ L
HotScript M-MuLV RTase (200U/ μ L) 0.8-1 μ L (see note 4)
RNase free H2O to 20 μ L
Gently mix well
If Oligo (dT) 18 or gene specific primers (GSP) are used, incubate at 50 ℃ to 55 ℃ for 30-50 minutes.
If using Random Primer, incubate at 23 ℃ for 10 minutes and 50 ℃ to 55 ℃ for 30 to 50 minutes.
Deactivate HotScript M-MuLV RTase by heating at 85 ℃ for 5 minutes.
Note: High GC content (>50%) or secondary
