台灣轉錄科技有限公司     分生器材網

Two Step RT-PCR

產地：丹麥

Cat. No.: 761004 (1000 Units)

Description

Avian Myeloblastosis Virus (AMV) reverse transcriptase is an RNA- dependent DNA Polymerase that uses single stranded RNA or DNA as a template to synthesize the complementary  DNA strand. AMV-RT is suitable for synthesizing long cDNA transcript and for dideoxysequencing of DNA and RNA. The AMV Reverse Transcriptase is isolated from Avian Myeloblastosis Virus particles.

Ampliqon Taq DNA Polymerase is a thermostable recombinant DNA polymerase, which exhibits very high activity in primer extension and other molecular biology applications. The enzyme is isolated from Thermus aquaticus and has a molecular weight of approximately 94 kDa.

Ampliqon Taq DNA Polymerase has both a 5' to 3' DNA polymerase and a 5' to 3' exonuclease activity. The enzyme lacks a 3' to 5' exonuclease activity (no proofreading ability). Taq DNA Polymerase leaves an A’ overhang, which makes the enzyme ideal for TA cloning.

Store at -20°C. Reagent for in-vitro laboratory use only

Kit Components Volume

AMV Enzyme 50 μL

10x AMV Reaction Buffer 1.5 mL

Taq DNA Polymerase 100 μL

10x Ammonium Buffer (MgCl_{2 15mM) 1.5 mL}

AMV Reverse Transcriptase Activity Assay

First-Strand cDNA Synthesis

First-strand cDNAs of 1.2kb and 7.4kb control RNAs are synthesized at 37°C for 1 hour using 15 units of AMV Reverse Transcriptase, 1μg of each template, an oligo(dT)_{15 primer and radiolabeled dCTP. The minimum specification is the production of 150ng of first-strand cDNA. Full-length cDNA for the 1.2kb RNA must be observed by gel electrophoresis and autoradiography.}

AMV Reverse Transcriptase Contaminant Activity

DNase and RNase Assay

For test of nuclease activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 15 units of AMV Reverse Transcriptase in 1X AMV Reaction Buffer for one hour at 37°C, and the release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material.

Endonuclease Assay

For test of endonuclease activity, 1μg of Type I supercoiled plasmid DNA is incubated with 15 Units of AMV Reverse Transcriptase in 1X AMV Reaction Buffer for one hour at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.

Notes:

1. Always wear gloves when handling RNA to avoid contamination with RNases.
2. Use 5 U AMV Reverse Transcriptase Enzyme per μg polyA+ RNA (mRNA).
3. Use 10 U AMV Reverse Transcriptase Enzyme per μg Total RNA.
4. Use 0.5μg Random Hexamer or Oligo(dT)15-25 primer/μg RNA
5. Use 10-20 pmol Gene-Specific primer/μg RNA
6. Use of Gene-Specific Primer synthesizes a specific transcript.
7. Use of Random Hexamer; Synthesize random transcripts with different length and at different location.
8. Use of Oligo(dT)15-25; Synthesize multiple fragments of different length from the genes 3?end.

Protocol for First-Strand cDNA Synthesis

The following procedure uses 2μg of RNA.

2. Heat the tube to 70°C for 5 minutes to melt secondary structure within the template. Cool the tube immediately on ice to prevent secondary structure from reforming, and then spin briefly to collect the solution at the bottom of the tube.

3. Add the following components to the annealed primer/template in the order shown for a final of 25μl reaction volume.

    2.5μL 10X AMV Reaction Buffer

    2.0μL dNTP Mix (12.5mM)

    1.0μL RNA Safe (40 U/μL) (OPTIONAL)

    20 U AMV RT Enzyme

    Add Nuclease free water to 25μl

4. Mix gently by flicking the tube and incubate for 60 minutes at 37°C for random primers or 42°C for other primers or primer-adaptors.

5. Before proceeding to PCR, inactivate the AMV Reverse Transcriptase by heating to 70^{OC for 15 min.}

^{Suggested Protocol using Taq DNA Polymerase}

Table 1. Using X μL of First-Strand Synthesize correspond to X mM MgCl_{2 in a 50 μL PCR reaction}

Notes:

1. 15 mM MgCl_{2 is present in the 10X Ammonium buffer. The 1X concentration is 1.5 mM MgCl2.}
2. In some applications, more than 1.5 mM MgCl2 is needed for the best results. For this reason, 25 mM MgCl2 is included with the kit. Table 3 provides the volume of 25 mM MgCl2 to add to the master mix if a higher MgCl2 concentration is required. Remember the carry-over amount of MgCl2 from the first-strand synthesis.

Table 2. PCR Reaction components

Table 3. MgCl_{2 concentration in a 50 μL reaction}

2. Mix the master mix thoroughly and dispense appropriate volumes into reaction tubes. Mix gently, e.g., by pipetting the master mix up and down a few times.

3. Add template DNA to the individual tubes containing the master mix.

7-5542589 ; 0800 231 233