A Quantity Correction Method for Plasmid DNA Molecular Standard Substance
A technology of DNA molecules and standard substances, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of plasmid DNA molecular weight bias and other problems, and achieve good stability and accurate quantitative results
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Embodiment 1
[0027] Genomic DNA extraction and detection
[0028] 1. Plant genomic DNA extraction:
[0029] The kit DP305 (Tiangen Biochemical Technology Co., Ltd.) was used to extract and purify the genomic DNA of rice, soybean, corn, cotton, and rapeseed. The extraction method is as follows:
[0030] 1) Take 200 mg sample powder sample;
[0031] 2) Add 800 μL of 65°C preheated GP1 buffer, quickly invert and mix well, place the centrifuge tube in a 65°C water bath for 1 hour, and invert the centrifuge tube to mix the samples several times during the water bath.
[0032] 3) Add an equal volume of phenol:chloroform (1:1), mix well, and centrifuge at 12000rpm for 10min.
[0033] 4) Transfer the supernatant to a new centrifuge tube, add an equal volume of chloroform, mix thoroughly, and centrifuge at 12000 rpm for 10 min.
[0034] 5) Take the supernatant, add an equal volume of GP2, and mix well.
[0035] 6) Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000 rp...
Embodiment 2
[0055] digital PCR
[0056] Dilute the DNA extracted from the leaves of each species to 1ng / μL as a spare background DNA solution, and then use the diluted background DNA solution to make the plasmid copy number 5.84×10 9 Copies / ul of GA21 and copy number 6.34×10 9 copies / μL of GTS were serially diluted, and the final copy number was 5.84×10 4 copies / μL and 6.34×10 4 copies / μL. Plasmids GA21 and GTS were assessed using the same digital PCR assessment method. In the digital PCR system, 1 μL was used as a template. Transformant-specific quantitative primers and probes were designed according to the GA21 and GTS inserted into the gene vector and the junction region sequence of its 5' side sequence as the target sequence, and adh1 and lectin were used as endogenous reference genes. Quantitative primers and probes are listed in Table 1.
[0057] Table 1 Digital PCR detection primers and probes
[0058]
[0059] Set GA21, GTS digital PCR reaction system as shown in Table 2-...
Embodiment 3
[0074] Results collection and statistics
[0075] Plasmid GA21 and GTS theoretical values:
[0076] In GA21 and GTS plasmids, the exogenous detection fragment and endogenous gene are 1:1, that is to say, each plasmid constructs 1 exogenous detection fragment and 1 endogenous gene on the plasmid vector. According to this, after microdroplet digital PCR amplifies the plasmid, the theory should be 1:1, which can also be expressed as exogenous / endogenous = 1.00.
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