StartScript® II 1st Strand cDNA Synthesis Kit,specification,price,image-Bio-Equip in China

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Product Specification

Physical Appearance	Liquid
Stability & Storage	Store at -25 ~ -15℃ for 2 years
Reference	[1] Roth, M J , N. Tanese , and S. P. Goff . "Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. " Journal of Biological Chemistry 260.16(1985):9326. [2] Kotewicz, Michael L , et al. "Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity." Nucleic Acids Research 16.1(1988):265-277.

Background

The StartScript II cDNA First Strand Synthesis Kit contains two optimized mixes: StartScript II Enzyme Mix and StartScript II Reaction Mix. The enzyme mixture contains StartScript II reverse transcriptase and mouse RNase inhibitor; the reaction mixture contains dNTP and optimized buffer. StartScript II reverse transcriptase is a recombinant M-MLV reverse transcriptase that reduces RNase H activity and improves thermal stability. StartScript II reverse transcriptase synthesizes first-strand cDNA at higher temperatures compared to wild-type M-MLV reverse transcriptase. The enzyme has an activity temperature of up to 48°C and has the characteristics of high specificity and high cDNA yield.

The kit also offers two optimized reverse transcription primers with nuclease-free water contaminated water. The Oligo-dT primer [d(T) 23 VN] can anneale with the start of the polyA tail. The optimized random primer mix can be randomly and continuously paired with the entire RNA template, including mRNA and polyA-free RNA. The synthesized first-strand cDNA product can be up to 10 kb in length.

Components

组分	UA070076-50 Rxns	UA070076-250 Rxns
StartScript II Reaction Mix (2X)	600 μl	5 x 600 μl
StartScript II Enzyme Mix (10X)	100 μl	5 x 100 μl
Random Primer Mix (60 μM)	110 μl	5 x110 μl
Oligo(dT)23 VN* (50 μM)	110 μl	5 x 110 μl
RNase Free dH2O	1 ml	5 x 1 ml

* V = A, G or C; N = A, G, C or T

Protocol

If denaturation of template RNA is desired, use the following protocol.

1. Mix RNA sample and primer in a sterile RNase-free microfuge tube.

Number	Component	Volume
1. Template RNA (One of the three is optional)	Total RNA Or poly(A) RNA/mRNA Or specific RNA	< 5 μg < 1 μg < 0.5 μg
2. Primers (One of the three is optional)	Oligo(dT) 23 VN (50 μM) Or Random Primer Mix (60 μM) Or Specific Primer (1 μM)	2 µl
3. H2O	RNase Free dH2O	Up to 8 μl

2. Denature sample RNA/primer for 5 minutes at 65°C. Spin briefly and put promptly on ice.

3. Add the following components

StartScript II Reaction Mix (2X)	10 μl
StartScript II Enzyme Mix (10X)	2 μl

4. Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If Random Primer Mix is used, an incubation step at 25°C for 5 minutes is recommended before the 42°C incubation.

5. Inactivate the enzyme at 80°C for 5 minutes. The cDNA product should be stored at –20°C. In general, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume.

Guidelines

1. Use DEPC to dispose of all equipment used in the study, or purchase equipment that has been proved to be nucleic acid-free. Wear gloves during the study and change them frequently to avoid RNA enzyme contamination.

2. Ensure that there is no RNA enzyme contamination in the reagents used.

3. In order to ensure the effective retrotranscriptional reaction, it is necessary to use high-quality RNA templates.

4. Poly (A)+ mRNA does not need to be isolated from total RNA, but using Poly (A)+ mRNA as a template can improve the yield and purity of the final product. The amount of RNA used, the total RNA is less than 5 μg; Poly (A)+ RNA is less than 1 μg; Specific RNA is less than 0.5 μg.

5. The cDNA products should be stored at -20°C.

6. Enzymes should be placed on ice during experimental operation, and stored at -20°C after the experiment.