Methods of amplifying mRNA and making full-length mRNA libraries
A full-length, sample technology, applied in the field of reagent kits and genetic diagnosis, which can solve the problem of lack of new solutions and wide commercial applications.
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Embodiment 1
[0127] Embodiment 1 (with image 3 relevant)
[0128] For internal controls, in addition to housekeeping genes like GAPDH, eGFP (Baseclick), eGFP (Baseclick), Cas9 (Trilink), β-Gal(Trilink) and Fluc (Trilink) mRNA (0.1 μg). Add 1 μL of dNTP mix (10 mM) and 2 μL of Poly(dT) primer (100 μM)) to this RNA pool and adjust to a total volume of 13 μL with RNase-free HO. The mixture was incubated at 65°C for 5 min, cooled to 0°C for 3 min to allow hybridization. For cDNA synthesis, add 4 μL of 5x SuperScriptIV buffer, 1 μL of dithiothreitol (100 mM), 200 units of Superscript IV reverse transcriptase, and fill up to a total volume of 20 μL with RNase-free water. The mixture was incubated at 50°C for 20 minutes, at 80°C for 10 minutes, and cooled to 4°C for 3 minutes. After cDNA synthesis add 3 μL 10x RNase H buffer, 1 μL RNase A (10 mg / mL), 1.4 μL RNase H (5 U / μL) and 4 μL shrimp alkaline phosphatase (1 U / μL) and 0.6 μL dH 2 O to remove RNA and excess nucleotides. The mixtur...
Embodiment 2
[0140] Embodiment 2 (with Figure 4 relevant)
[0141] After click ligation (procedure described in Example 3), specific genes were amplified from the cDNA library using targeting primers (various reverse and forward Intern primers, see table below), which generated specific PCR fragments. In a 200 μL reaction vial, mix 11.5 μL dHO 2 O, 4 μL 5x OneTaq standard reaction buffer, 1 μL Intern reverse primer (10 μM), 1 μL forward Intern primer (10 M), (10 μM), 0.4 μL dNTP mix (10 mM), 2 μL purified and click-ligated cDNA mix, and 0.13 μL OneTaq DNA polymerase (5000 U / μL) (New England Biolabs) was mixed. Samples were subjected to a thermal cycling program in a thermal cycler (BioRad).
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[0144] A 5 [mu]L unpurified aliquot of each PCR amplification was analyzed on a 1.5% agarose gel (10x15 cm) prepared in TAE buffer (20 mM TRIS, 10 mM acetic acid, 0.5 mM EDTA).
[0145] Oligonucleotides:
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Embodiment 3
[0148] Embodiment 3 (with Figure 5 with Figure 12 relevant)
[0149] Click to connect ( image 3 After the procedure described in the Examples), specific genes were amplified from the cDNA library using gene-specific primers and common adapter primers targeting primers (various Intern reverse primers and adapter primer 1, see table below), which generated Specific PCR fragments. Mix 11.5 µL dH in a 200 µL reaction vial 2 O, 4 µL 5x OneTaq standard reaction buffer, 1 µL Intern reverse primer (10 µM), 1 µL Adapter primer 1 (10 µM), 0.4 µL dNTP mix (10 mM), 2 µL purified and click-ligated cDNA mix, and 0.13 µL OneTaq DNA Polymerase (5000 U / μL) (New England Biolabs). Samples were subjected to a thermal cycling program in a thermal cycler (BioRad).
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[0151] A 5 [mu]L unpurified aliquot of each PCR amplification was analyzed on a 1.5% agarose gel (10 x 15 cm) prepared in TAE buffer (20 mM TRIS, 10 mM acetic acid, 0.5 mM EDTA).
[0152] for Figure 12 For Sanger...
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