A positive plasmid molecule pbi121-screening and its application
A positive plasmid and molecule technology, applied in the direction of using vectors to introduce foreign genetic material, microbial assay/inspection, recombinant DNA technology, etc., can solve the reports that the development and application of positive plasmid molecules for screening and detection of genetically modified crops have not been found, and no consideration has been given to them. The presence or absence of screening elements, the ratio of copy numbers, differences in extraction efficiency, etc.
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[0098] Below in conjunction with accompanying drawing and embodiment the present invention is described in detail:
[0099] 1. Construction of positive plasmid molecule pBI121-Screening
[0100] 1.1. Genomic DNA extraction from transgenic materials
[0101] First preheat 20% SDS to 65°C, take 15ml of SDS extraction buffer (0.1TrisHCl, 0.05MEDTA, 1MNaClpH8.0) into a 50ml centrifuge tube, then add 2.5μl of β-mercaptoethanol, mix well; liquid nitrogen grinds about 3g Leaves, transfer the powder to 50ml of 50ml centrifuge tube containing extraction buffer, shake and mix on the shaker, add 2ml of preheated 20% SDS, mix well, 65 ℃ water bath for at least 30 minutes, during which the test tube should be shaken gently ;After the water bath, quickly place the centrifuge tube on ice, add 3ml 3MKAc, mix well, and place on ice for 30 minutes; centrifuge at 5000g at 4°C for 5min; transfer the supernatant to a new 50ml centrifuge tube, add 2 / 3 volume of Isopropanol, mix well, place at -20...
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