EzSCRIPT™ III RH 5X Master Mix (RH, 5x, MMLV-RT, RNase H⁺, V2)

CAT #EBT-1541 / #EBT-1541-5

QTY 0.2 ml (50 rxn / 4ul)

Description

Elpis Biotech’s Reverse Transcription Master Premix is a mixture of M-MLV reverse transcriptase (RNase H+ ), dNTPs, reaction buffer, and primers (random hexamer (EBT-1541) or oligo d(T)15 primer (EBT-1542)) for the first strand cDNA synthesis. Users can perform reverse transcription by simply adding total RNA or poly(A) RNA sample to the premix. Reverse Transcription Master Premixes are supplied as a 5x stock solution. Stabilizer in premix enables prolonging a shelf life at -20℃. If you want to use your own primer pair for cDNA synthesis, you can use the product, EBT-1543, which do not contain any cDNA synthesis primers. M-MLV-RTase (RNase H+, Ver2) is recommended for specific cDNA synthesis reducing artifact by RNA secondary structure and is suitable for One-step RT-PCR.

Applications

First strand cDNA synthesis from total RNA or polyA+ RNA, Primer extension, RT-PCR.

QC tests

- Performance tests.

Storage Condition

- Store at -20℃ for one year.

Usage Information (protocol)

1. Add 1 ㎍ total or 100 ng polyA+ RNA sample in a total volume of ≤16 ㎕ in nuclease-free water.

2. Heat the tube to 70 ℃ for 5min to melt secondary structure within the RNA template.

3. Cool the tube immediately on ice, then spin briefly to collect the solution at the bottom of the tube.

4. Add the followings.

Reverse Transcription Master Mix 4 ㎕

Denatured RNA sample 16 ㎕

5. Mix gently by flicking the tube, and incubate for 30 min at 37 ℃ ~ 65 ℃.

6. To stop reaction, incubate for 5min at 94 ℃.

Notes

The cDNA by reverse transcription can be used for subsequent PCR reactions and for the cDNA library constructions. If there is concern about possible RNase contamination in the reaction, Recombinant RNase Inhibitor may be added to the reaction to preserve RNA integrity.

PCR

1. Add the following components to the PCR tubes (20 ㎕ total reaction).

2. Perform PCR reaction as follows