ODN-X (2 μL, 4 μM) was mixed with 1 μL of 2 μM 12 nt RT primer 5′-HEX-AGAATCATCGAA and denatured at 70 °C for 5 min, then cooled down slowly to 4 °C (1 °C per 10 s) and hold at 4 °C. After adding 5 U M-MuLV reverse transcriptase (NEB) or 5 U SS III reverse transcriptase (Invitrogen), 2 μL corresponding 5 × buffer (NEB M-MuLV buffer or 5 × FS buffer), 3 μL of 100 μM dNTPs and to the RNA-primer mix (For ODN-Tm6 (link)A, additional 1 μL of 100 mM DTT was added), the RT reactions were carried out at 42 °C for 30 min. Then 30 μL deionized formamide was immediately added to the reaction mixture and heated to 90 °C for 10 min, followed by 20% denaturing PAGE analysis.
M mulv buffer
Manufactured by New England Biolabs
M-MuLV buffer is a solution used in reverse transcription reactions to facilitate the conversion of RNA to cDNA. The buffer provides the necessary ionic conditions and cofactors required for the optimal activity of the M-MuLV reverse transcriptase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
M mulv reverse transcriptase, by New England Biolabs (3 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ssiii reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
Recombinant rnasin, by Promega (1 mentions)
Dntps, by New England Biolabs (1 mentions)
Maxima reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Ribolock rnase inhibitor, by New England Biolabs (1 mentions)
Phasemaker tube, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
Random primer 9, by New England Biolabs (1 mentions)
Protector rnase inhibitor, by Roche (1 mentions)
4 protocols using m mulv buffer
1
RT-PCR of ODN-Tm6 (link)A
2
Amplified Probe Set Synthesis and Labeling
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The amplified probe set was in vitro transcribed using a HiScribe T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, E2050S). Each probe set was prepared as follows: 10 μl PCR product, 10 μl NTP buffer mix (from HiScribe kit), 2 μl T7 Polymerase mix (from HiScribe kit), 0.5 μl Recombinant RNasin (Promega, N2511), and 7.5 μl ddH2O. The samples were incubated at 37°C in the PCR machine for 4 h. For reverse transcription and fluorophore attachment, M‐MuLV Reverse Transcriptase (New England BioLabs, M0253L) was used. Each probe was prepared as follows: 7 μl dNTPs (New England BioLabs, N0447S), 7 μl 10× M‐MuLV Buffer, 10 μl of 100 μM A594‐labeled forward primer, 1.2 μl M‐MuLV enzyme, 1.4 μl Recombinant RNasin, 13.4 μl nuclease‐free water, and 30 μl RNA from the T7 reaction. The reaction mix was incubated at 50°C for 2 h.
3
Single-cell cDNA Synthesis Protocol
4
Zebrafish Embryo and Tissue RNA Extraction and cDNA Synthesis
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Each total RNA sample was extracted from 30-50 zebrafish embryos, single embryos or adult tissue samples by homogenizing them in 500μL Trizol reagent (Thermo Fisher Scientific, 15596026) using 1mL syringe and 21G needle and RNA was purified from lysates according to the Phasemaker tubes protocol (Thermo Fisher Scientific, A33248). For cDNA synthesis, a 4-μg aliquot of total RNA was treated with TurboDNAse using the TurboDNA-free kit (Thermo Fisher Scientific, AM1907). cDNA was produced by mixing 10μL of DNAsetreated RNA with 4μL of 2.5 mM dNTP and 2 μL of 80μM Random Primer 9 (NEB, S1254S) or 100μM oligo-dT(15-18) (Integrated DNA Technologies), heating at 70°C for 10 min and cooling on ice. Subsequently, 2μL of M-MuLV buffer (NEB, M0253S), 0.25μL of Protector RNAse Inhibitor (Roche, 03335399001), 0.25μL of M-MuLV reverse transcriptase (NEB, M0253S) and 1.6μL of water were added followed by incubation at 42°C for 1 hour and 10 min at 90°C. To amplify p53 cDNA fragments, we used p53cDNA primers (S1 Table ) to run the PCR on odT-based cDNA using Q5 High-Fidelity 2X Master Mix (NEB, M0492) with its standard program and annealing temperature of 64°C.
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