dART is modified RNA dependent DNA polymerase, that synthesizes a complementary DNA strand from a single-stranded RNA in the presence of reverse primer.
Kit is designed to amplify DNA from any RNA with high specificity and sensitivity in a two-step process.cDNA synthesis is performed in the first step using either total RNA or poly(A)+-RNA primed with oligo(dT), random hexamers primers or reverse gene specific primer. The second step, in separate tube is PCR where cDNA is a template and specific primers are used to amplify double-stranded DNA of interest using high fidelity OptiTaq DNA Polymerase.
Description:
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Reduced RNase H activity.
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Synthesizes single-stranded DNA on RNA template in a broad range of temperatures from 35°C to 55°C.
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Can be used for preparing labeled hybridization probes.
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Ideal for amplifying whole genes for cloning and sequencing.
Unit Definition: One unit is the amount of enzyme required to incorporate 1 nmol of dTTP into acid-insoluble form in 10 min at 37oC (1).
Storage Conditions: Store at -20°C
Quality Control: All preparations are assayed for contaminating endonuclease and exonuclease and nonspecific RNase and single- and double-stranded DNase activities.
References:
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Houts, G.E., Masakau, M., Ellis, C., Beard, D. and Beard,J.W. (1979) J. Virol. 29, 517-522.
Reagents are provided for 25 or 100 cDNA synthesis reactions of 20 μl each.
Component: 25 rxn kit: 100 rxn kit:
dART Revrese Transcriptase 25 μl 100 μl
5 x cDNA Buffer 150 μl 600 μl
100 mM DTT 50 μl 200 μl
5 mM dNTPs mix 150 μl 4 x 150 μl
RNase Inhibitor (12.5 U/μl) 25 μl 100 μl
Oligo(dT)20 (50 μM) 25 μl 100 μl
Random hexamers (50 ng/μl) 25 μl 100 μl
RNase-free Water 1.0 ml 4 x 1.0 ml
E.coli RNase H (2 U/μl) 25 μl 100 μl
Reagents are provided for 25 or 100 PCR reactions of 50 μl each.
Component: 25 rxn kit: 100 rxn kit:
OptiTaq DNA Polymerase 2.5 U/ μl 25 μl 100 μl
10 x Pol Buffer C with MgCl2 1.0 ml 4 x 1.0 ml
5 mM dNTPs mix 50 μl 200 μl
cDNA synthesis:
1. In 0.2-0.5-ml tube, combine primer (50 μM Oligo(dT)20, 50 ng/μl random hexamer primer or 10 μM reverse gene specific primer), RNA and dNPs mix and adjust volume to 13 μl with RNase free water.
Component: Amount:
primer 1 μl
RNA (10 ng-5 μg) x μl
5 mM dNTPs mix 4 μl
RNase-free Water to 13 μl
2. Heat RNA mix 5 min at 65°C and chill on ice for another 5 min.*
3. Vortex the 5 x cDNA Buffer.
4. Prepare a master reaction mix on ice and mix gently by pipeting on ice.
Component: Amount:
5 x cDNA Buffer 4 μl
RNase Inhibitor 12.5 U/μl 1 μl
100 mM DTT 1 μl
dART Reverse Transcriptase 1 μl
7 μl
5. Pipet 7 μl of master reaction mix into the 13 μl RNA mix kept on ice.
6.Transfer the sample to preheated to appropriate temperature thermal cycler. Incubate as follows:
Oligo(dT)20 primed : 30-60 min at 50°C (or 35-55°C)
Gene specific primed: 30-60 min at 50°C (or 35-55°C)
Random hexamer primed: 25°C for 10 min, followed by 20-50 min at 50°C (or 35-55°C).
7. Terminate the reaction by incubating at 85°C for 5 min.
8. Add 1 μl of RNase H and incubate at 37°C for 20 min (optional).
9. cDNA is ready for PCR, can be used immediately or stored at -20°C.
*- Heating step is an option. Applied in case of difficult RNA templates or strong secondary structures can improve results greatly. For all oter templates, heating step does not change reaction efficiency and could be avoided.
PCR with OptiTaq DNA Polymerase:
Use 2-5 μl of the cDNA as a template for PCR. The final magnesium concentration is 1.5 mM in reaction and in some cases there is a need of titration of magnesium to obtain best results.
1. Mix as follows all reagents in 0.2-0.5-ml tube.
Component: Amount:
cDNA template 2-5 μl
10 x Pol Buffer C 5 μl
dNTPs mix 5 mM each 2 μl
10 μM sense primer 1 μl
10 μM reverse primer 1 μl
OptiTaq DNA Polymerase 2.5 U/ μl 1 μl
RNase-free Water to 50 μl
2. Mix gently by pipeting.
3. Incubate at 94°C for 3 min, then perform 20-40 cycles of PCR with optimized conditions for Your sample (1 min/kb extension time at 68-72°C).
4. Analyze 10-20 μl of PCR sample by agarose gel electrophoresis.