Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified cotton
A technology of PCR-DHPLC and detection method, which is applied in the field of multiple PCR-DHPLC detection primers and detection of transgenic cotton, and achieves the effects of good expansion performance, high sensitivity and resolution, and easy operation
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Embodiment 1D
[0043] Example 1DNA Extraction
[0044] Sample DNA was extracted by CTAB method, as follows:
[0045] a) Weigh 5 g of the sample, add liquid nitrogen to the mortar and grind until the sample is a powder with a size of about 0.5 mm;
[0046] b) Weigh 300 mg of the ground sample, quickly transfer it to a 2 mL centrifuge tube, add 700 μL of CTAB extract solution preheated at 65 °C, mix well, and put it in a water bath at 65 °C for 30 min;
[0047] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;
[0048] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;
[0049] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;
[0050] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;
[0051] g) Add an equal volume of pre-cooled isopropanol, shake gently, ...
Embodiment 2D
[0054] Example 2 DNA concentration determination
[0055] The concentration and purity of the extracted sample DNA were measured; the absorbance values at 260nm and 280nm were measured by an ultraviolet spectrophotometer, and the purity and concentration of nucleic acid were calculated respectively. The calculation formula is as follows:
[0056] DNA purity = OD260 / OD280
[0057] DNA concentration=50×OD260mg / mL
[0058] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.
Embodiment 3
[0059] Embodiment 3PCR amplification
[0060] According to the sad I gene, transgenic lines MON531 and MON15985 design the binding site of the specific detection primer, design the specific primer binding site of the strain according to the transgenic line MON1445, and add the regulatory sequence at the 5' end of the above primer binding site, Synthesize detection primer pairs containing regulatory sequences (Table 1).
[0061] Table 1 Transgenic cotton detection primer binding sites and primers
[0062]
[0063] The total volume of the PCR reaction system is 50 μL, and the components are: multiplex PCR reaction mixture Multiplex PCR Mix (TaKaRa) 25 μL, 10 μmol / L primers 1 μL, DNA 2 μL, 5U / μL Taq enzyme 0.25 μL, with sterilized double distilled water Make up to 50 μL.
[0064] PCR reaction conditions: denaturation at 94°C for 1min; then 35 cycles of 94°C for 30s, 57°C for 1min, and 72°C for 1min; after the cycle, extend at 72°C for 10min.
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