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RT-PCR, Technical Appendix
Determination of concentration and purity of RNA
Concentration of RNA
The concentration of RNA may be determined spectrophotometrically at 260 nm, where 1 absorbance unit (A260) = 40 µg of single-stranded RNA/ml.
Purity of RNA
The ratio of absorbances at 260 nm and 280 nm (A260/A280) provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV region, such as proteins. Pure RNA should exhibit an A260/A280 ratio of 1.9-2.1 in 10 mM Tris-Cl, pH 7.5. The A260/A280 ratio is influenced by pH and since water is not buffered, it is recommended to measure the absorbance of RNA sample in 10 mM Tris-Cl, pH 7.5.
Storage of RNA
Purified RNA may be stored at -20 °C or -70 °C in water. For long term storage, ethanol precipitation of the RNA is recommended; add sodium acetate, pH 4.0, to 0.25M, and 2.5 volumes of 100 % ethanol. Store at -70 °C.
Specification of RobusT™ I RT-PCR Kit Components
(F-580S, F-580L)
AMV Reverse Transcriptase (RobusT I RT-PCR Kits, F-580S and F-580L)
The AMV Reverse Transcriptase, isolated from Avian Myeloblastosis Virus, is used for first strand cDNA synthesis. The RT step can be performed at elevated temperatures up to 60 °C. High temperature increases the specificity of priming and helps to increase the efficiency by reducing RNA secondary structures.
Concentration: 5 U/µl
Storage Buffer: 0.2 M KPO4, pH 7.2, 2.0 mM DTT, 0.2 % Triton® X-100, 50 % glycerol (v/v).
DyNAzyme™ EXT DNA Polymerase
In the RobusT RT-PCR Kit second strand cDNA synthesis and high fidelity PCR amplification of cDNA are performed by DyNAzyme™ EXT DNA Polymerase, which is a versatile and easy-to-use enzyme with powerful advantages for all PCR applications. DyNAzyme EXT DNA Polymerase is an optimal mixture of recombinant DyNAzyme™ II DNA Polymerase and a proofreading enzyme. DyNAzyme EXT DNA Polymerase contains sufficient amounts of proofreading activity to remove misincorporated nucleotides. Thermostable DNA polymerases without proofreading activity are incapable of removing misincorporated nucleotides and thus in most cases are incapable of continuing the polymerization. This reduces the amount of final product formed. For this reason a low amount of proofreading activity is needed to remove misincorporations that could terminate DNA synthesis. DyNAzyme EXT DNA Polymerase exhibits fidelity comparable to the best of traditional cloning enzymes yet providing a much broader working range.
Concentration: 1 U/µl
Storage Buffer: 20 mM Tris-HCl (pH 7.4 at 25 °C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5 % Tween® 20, 0.5 % Nonidet P 40, 200 µg/ml BSA, 50 % glycerol (v/v)
10x RobusT™ Reaction Buffer
RobusT I RT-PCR Kit is supplied with a 10x RobusT Reaction Buffer, which is specifically optimized for RobusT RT-PCR Kit reactions.
50 mM MgCl2 solution
dNTP mix
Concentration: 10 mM of each
The dNTP Mix is a ready-to-use solution consisting of the following compounds: dATP, dGTP, dCTP and dTTP. The deoxynucleosidetriphosphates are dissolved in water, pH 7.0.
dATP, 2´-DEOXYADENOSINE 5´-TRIPHOSPHATE,
minimal diphosphate, sodium salt
C10H12N5O12P3Na4 F. W. 579.2
dGTP, 2´-DEOXYGUANOSINE 5´-TRIPHOSPHATE,
minimal diphosphate, sodium salt
C10H12N5O13P3Na4 F. W. 595.1
dCTP, 2´-DEOXYCYTIDINE 5´-TRIPHOSPHATE,
minimal diphosphate, sodium salt
C9H12N3O13P3Na4 F. W. 555.1
dTTP, 2´-DEOXYTHYMIDINE 5´-TRIPHOSPHATE,
minimal diphosphate, sodium salt
C10H13N2O14P3Na4 F. W. 570.1
Upstream control primer
20 µl 10 pmol/µl in dd water
5´-GGCCGGCAGGTGGTTGGAGT-3´
Tm 74.3 °C
Downstream control primer
20 ml 10 pmol/ml in dd water
5´-GGAGTTTGCTGCGATTGCTGAGG-3´
Tm 72.4 °C
Control RNA with carrier
20 µl 10 ng MS2 RNA/µl
30 µg E. coli ribosomal RNA/ml in dd water
Quality control
The product is tested functionally using 1 pg of MS2 RNA. Template RNA can be replicated and amplified into a clear, discrete 1011 bp DNA product.
