Color reverse transcription premixed liquid
A technology of reverse transcription and premix solution, which is applied in the field of molecular biology and can solve the problems such as the problem of reaction system preparation not being effectively solved and so on.
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Embodiment 1
[0023] Embodiment 1, the preparation of reverse transcription master mix
[0024] The components necessary for reverse transcription are: reverse transcriptase (MMLV or AMV reverse transcriptase), magnesium salts (such as magnesium chloride, magnesium sulfate, etc.), potassium salts (potassium chloride, potassium sulfate, etc.), buffer components (such as Tris-HCl, Bis-Tris, HEPES, Tricine, TAPS, etc.), dNTPs (including dATP, dTTP, dCTP and dGTP) and primers (such as Oligo-dT and random hexamer primers), etc. In order to prepare the reverse transcription master mix, in addition to optimizing the concentration of these necessary components, in order to ensure the stability and reaction performance of the master mix, it is also necessary to add some other components, such as RNase inhibitor (RNase inhibitor, RI), glycerol , DTT, one or more of surfactants (such as Triton, Tween, etc.), and optimize them.
[0025] components concentration Tris-HCl (pH8.5) 100mm...
Embodiment 2
[0027] Embodiment 2, the preparation of color reverse transcription master mix
[0028] Although there are many kinds of dyes commonly used in biological experiments, they are not necessarily suitable for the preparation of color reverse transcription master mixes. Because the dye added to the master mix is always present throughout the reverse transcription-real-time quantitative PCR experiment, several factors should be considered: first, the presence of the dye cannot interfere with the reverse transcription reaction, nor can it affect the reverse transcription reaction. Enzyme activity and stability; secondly, in general, the cDNA obtained by reverse transcription needs to be diluted before it can be used as a template for real-time quantitative PCR, so the color of the dye should be bright enough, otherwise it will be difficult to prepare the real-time quantitative PCR reaction system. It cannot be easily seen whether the cDNA template has been added; finally, the prese...
Embodiment 3
[0032]Embodiment 3, the use of color reverse transcription master mix
[0033] Using human 293T cell total RNA as a template, perform reverse transcription:
[0034] 1. First measure the RNA concentration, take 1 microgram of RNA, add water to 16 μL.
[0035] 2. Add 4 μL of color reverse transcription master mix, mix by pipetting and centrifuge briefly, and collect the liquid at the bottom of the tube.
[0036] 3. Incubate at 42°C for 15-60 minutes to obtain cDNA.
[0037] As can be seen from the above steps, using the color reverse transcription master mix, the experimenter only needs to calculate the volume of RNA template and water required for the experiment, and then add a fixed volume of color reverse transcription master mix. It is not necessary to mix components such as reverse transcriptase, primers, and reaction buffer in sequence in addition to the RNA template and water, as in the traditional reverse transcription method. Naturally, there is no need to calculate ...
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