2.4 Checking mRNA and its concentration

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2.4 Checking mRNA and its concentration

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The following procedure is for ethanol-purified products by neutralization with ammonium acetate.

Check and prepare 40 μl mRNA solution obtained after purification of transcription.

Please note there is an optimal amount of mRNA to add to the translation reaction.

Check mRNA concentration

Check if the concentration of 40 μl mRNA solution is within following range

mRNA concentration:　1500 ng/μl ± 400 ng/μl

If concentration is 1000 ng / μl or less, or 2000 ng / μ or more, one of PCR, transcription or purification may not have been performed correctly.

Check mRNA by electrophoresis

Confirm that a single band is clearly observed by electrophoresis of 1μl sample. If an extra band appears, PCR may not be performed correctly.サン

 Preparation of optimal mRNA amount

The optimum amount of mRNA for a minimum translation reaction volume of 110 μl is as follows.

optimum mRNA amount: 35 μg ± 5 μg

mRNA solution needed to be added in an 80 μl solution, so that when the mRNA concentration is X μg / μl, To make amount 35 μg, (35 / X) μl out of  40 μl needs to be filled up to 80 μl.

e.g. ）When concentration of mRNA is 1631 ng/μl, To get 35 μg of mRNA, 35  / 1.631 ≒　21 μl . Thus, get 21 μl out of 40 μl mRNA solution and fill up to 80 μl