Universal RT-PCR Kit(M-MLV)

This kit is suitable for reverse transcription reactions and subsequent PCR amplification of various RNA products. It uses M-MLV for retrotranscription reaction and can obtain longer retrotranscription products. At the same time, in the 20μL reverse transcription system and 50μL PCR reaction system, enough PCR products can be obtained at one time for subsequent cloning experiments. The enzymes formulated in this kit are all imported enzymes. RT enzyme uses imported M-MLV, so the cDNA is longer and the gene information is retained more completely! Specific RNase inhibitors during reverse transcription can effectively reduce the risk of experimental failure due to contamination by exogenous RNase. The kit is convenient and fast to use, and can be widely used in molecular biology experiments such as cDNA cloning and target gene detection.

General purpose RT-PCR Kit (M-MLV) product introduction

Moloni Mouse leukemia virus reverse transcriptase (M-MLV RT) is an RNA-dependent DNA polymerase that transcribed mRNA (>5kb) into cDNA. The RNase H activity of M-MLV RT is weaker than that of avian myeloblastoma virus reverse transcriptase, and it is more suitable for reverse transcription of longer mRNA. M-MLV RT is used to synthesize the first strand of cDNA using RNA as template.

Note: M-MLV RT is less extendable than AMV reverse transcriptase, so more units of M-MLV reverse transcriptase are needed to produce cDNA with the same yield as AMV reverse transcriptase

General RT-PCR kit (M-MLV) use method

1) Introduction to the use of 2mg total RNA as an example. In the RNase-free centrifuge tube, 0.5mg primer was added to the sample containing total RNA, with the total volume not greater than 15mL. The secondary structure formed by the template chain is opened at 70℃ for 5min. Immediately placed on ice to cool to prevent the secondary structure from recovering, followed by a brief centrifugation to concentrate the liquid at the bottom of the centrifuge tube.

2) Add the following ingredients to the annealed prime-template mixture.

Note: Do not change the insertion ratio of primers.

M-MLV 5×Reaction Buffer 5mL

dATP, 10mM 1.25mL

dCTP, 10mM 1.25mL

dGTP, 10mM 1.25mL

dTTP, 10mM, 1.25mL

Recombinant RNasin Ribonuclease Inhibitor 25units

M-MLV RT 200units

Nucleic acid-free water to final volume 25mL

3) Gently mix the centrifuge tube. If random primers are used, incubate at 37℃ for 60min. Other primers are incubated at 42℃. The optimum elongation temperature is between 37 ℃ and 42℃.

4) M-MLV RT is used to synthesize the first strand of cDNA. The subsequent synthesis needs to be synthesized again by designing primers according to the needs.

General purpose RT-PCR kit (M-MLV)Buffer composition

M-MLV RT 5×Reaction Buffer

250mM Tris-HCl (PH 8.3, at room temperature)

375mM KCl

15mM MgCl2

50mM DTT

Source of General purpose RT-PCR Kit (M-MLV)

E. coli expression and purification

General RT-PCR kit (M-MLV) storage conditions

Store at -20℃. Avoid constant freezing and thawing."

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.

Remark：These protocols are for reference only. Solarbio does not independently validate these methods.

Note：

1. The products are all for scientific research use only. Do not use it for medical, clinical diagnosis or treatment, food and cosmetics, etc. Do not store them in ordinary residential areas.

2. For your safety and health, please wear laboratory clothes, disposable gloves and masks.

3. The experimental results may be affected by many factors, after-sale service is limited to the product itself and does not involve other compensation.