Glucose Oxidase
2023-07-17
5x RT PCR MasterMix
2023-10-31
Product Number: RN02
Shipping and Storage
-20°C.
Description
Thermostable RNase H is an endonuclease that specifically hydrolyzes hybridization to DNA at high temperatures The phosphodiester bonds of the RNA on the strand, so it can degrade the RNA in the DNA/RNA hybrid strand, without digesting the single- or double-stranded DNA. Heat-resistant RNase H and RNase H in E. coli is a homologous protein and has endonuclease activity, but heat-resistant RNase H also exhibits activity at 65°C, which makes the enzyme Can be used for high temperature experiments. The enzyme gene is derived from the extreme thermophilus Thermus thermophilus, which is introduced into the E. coli plasmid and is expressed and purified.
Application
- Removal of mRNA that is heterozygous with the first strand of cDNA when synthesizing the second strand of cDNA;
- Remove mRNA poly(A) hybridized onto poly(dT);
- Lysis probe method to detect mRNA capping rate.
Notes
Since the reaction buffer contains Mg2+, under high temperature reaction conditions, when the system contains RNA/DNA heterozygous strands, there are other single-stranded RNAs, the temperature and reaction time should be appropriately reduced to reduce the impact on single-stranded RNA.
Components
| Components | volume |
| Thermostable RNase H (5U/μl) | 50μl |
| 10×Thermostable RNase H Reaction Buffer | 1ml |
Unit definition
At 45°C, in a 50μl reaction system,after 20 min the amount of enzyme required to be able to fully digest 1nmol [H3]-labeled Poly(rA) hybridized double-stranded RNA with Poly(dT) into ribonucleotides is defined as 1 active unit.
Quality control
After multiple column purifications, only clear single band of interest was visible for SDS-PAGE glue detection, and there was no E. coli DNA residue and no nucleic acid internal and external nuclease contamination by PCR method.
Protocol
1.Digestion of DNA/RNA heterozygous duplexes
| Components | volume |
| DNA/RNA heterozygous strands | 2μg |
| Thermostable RNase H | 5U |
| 10×Thermostable RNase H Reaction Buffer | 10μl |
| RNase Free Water | Up to 100μl |
Incubate at 45°C for 20 minutes, and after the reaction, 1μl of 0.5M EDTA can be used to terminate the reaction.
Lane1: DNA/RNA heterozygous double-stranded fragment is 810bp;
Lane2: DNA/RNA heterozygous double-strands are degraded by RNaseH and only single-stranded DNA remains.
2.Lysis probe method to detect mRNA capping rate
2.1 mRNA sample processing
Note: The following is an example of a biotin-labeled lysis probe.
| Components | volume |
| Biotin-labeled lysis probes | 500pmol |
| mRNA samples | 100pmol |
| Thermostable RNase H | 10μl |
| 10×Thermostable RNase H Reaction Buffer | 10μl |
| RNase Inhibitor, GMP Grade | 3μl |
| RNase Free Water | Up to 100μl |
The above mixture was mixed and placed in a PCR instrument, reacted at 50°C for 45min, and the lysis product was purified and tested on the machine.
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