Total RNA was isolated from strains PAO1 and PGW-ΔpsrA, and purified as described above. cDNA was synthesized in a 21 μl reaction volume from 5 μg of total RNA using a 1:1 mixture of GC-rich hexamers (Gene Link) and random hexamers (Invitrogen) for priming with 40 μM deoxynucleoside triphosphates (dNTPs) (USB). Reactions were then heated at 65°C for 5 min, followed by cooling to 4°C for 1 min. Following this step, 200 U of SuperScript III reverse transcriptase in First Strand buffer (Invitrogen) with 0.1 M dithiothreitol and RNase Out RNase inhibitor (Invitrogen) were added to each reaction mixture, yielding a final volume of 30 μl. Reaction mixtures were then heated to 25°C for 5 min, followed by 50°C for 1 hour and then 75°C for 15 min.
Superscript 3 reverse transcriptase in first strand buffer
Manufactured by Thermo Fisher Scientific
SuperScript III reverse transcriptase is an enzyme used for the synthesis of first-strand cDNA from an RNA template. It is provided in a First Strand buffer solution.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using superscript 3 reverse transcriptase in first strand buffer
1
RT-qPCR cDNA Synthesis Protocol
2
RNA Extraction and cDNA Synthesis
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