Molecular marker used for identification of Sanzan gum and synthesis bacteria thereof, and preparation and application of molecular marker
A technology of molecular markers and Sanzan glue, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome steps and achieve high accuracy and short detection time
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Embodiment 1
[0033] A molecular marker for identifying sanzan gum and its synthetic bacteria. The sequence of the primer 1 of the molecular marker is SEQ5: 5'-TCAGGCCGTGTGGGGAA-3', and the sequence of the primer 2 is SEQ6: 5'-GATCCGATCCAGCTTTCGGG- 3'.
[0034] The above-mentioned method for obtaining primers for identifying Sanzan gum and its synthetic bacteria, the steps are as follows:
[0035]1. The method for obtaining primers for identifying molecular markers of Sanzan Gum and its synthetic bacteria, the steps are as follows: Synthesize Sanzan Gum Synthetic Bacteria—Sphingomonas sp.CGMCC No.1650 and gellan gum Bacteria—Sphingomonas sp.CGMCC No.14238, Wenlun glue-synthesizing bacteria—Sphingomonas sp.CGMCC No.1561, Xanthan gum-synthesizing bacteria—Xanthomonas campestris Xanthomonas campestris CGMCCNo.10122 were respectively inoculated into 250mL Erlenmeyer flasks with 100mL sterilized liquid medium, and the medium formula was as follows (g / L): glucose 12.0, malt extract powder 4.0, p...
Embodiment 2
[0046] Detection of Molecular Markers of Sanzan Glue-Synthesizing Bacteria
[0047] Sanzan gum synthetic bacteria Sphingomonas sp.CGMCCNo.1650 as described in Example 1, gellan gum synthetic bacteria, Wenlun gum synthetic bacteria, and xanthan gum synthetic bacteria were respectively inoculated into 100mL sterile In a 250mL Erlenmeyer flask of bacterial liquid culture medium, the medium formula is as follows (g / L): glucose 12.0, malt extract powder 4.0, peptone 2.5, yeast powder 1.5, pH 7.0. 150r / min, 30°C shaker culture for 48h Afterwards, centrifuge at 6000r / min for 15min, collect the bacterial cells, and use the bacterial genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA. Use primer 1 and primer 2 as primers for PCR amplification. The PCR amplification system is: 1 μL of template DNA of 10-30 ng / uL, 0.5 μL of upstream and downstream primers, 1.0 μL of 10 mM dNTP, 2.5 μL of 10× buffer, 5 U / μL Platinum Taq DNA poly...
Embodiment 3
[0049] Inoculate Sanzan gum synthetic bacteria Sphingomonas sp.CGMCC No.19480 into a 250mL Erlenmeyer flask containing 100mL sterilized liquid medium. The medium formula is as follows (g / L): glucose 12.0, malt infusion Powder 4.0, peptone 2.5, yeast powder 1.5, pH 7.0. 150r / min, 30°C shaker culture for 48h, 6000r / min, centrifuge for 15min, collect bacterial cells, use Tiangen Biochemical Technology (Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit for genomic DNA extraction. Use primer 1 and primer 2 as primers for PCR amplification. The PCR amplification system is: 1 μL of 10-30ng / uL template DNA, 0.5 μL of upstream and downstream primers, 1.0 μL of 10 mM dNTP, 2.5 μL of 10× buffer, 5 U Add 0.5 μL of PlatinumTaq DNA polymerase / μL to 25 μL with water. The PCR reaction conditions are: 95°C pre-denaturation for 5 minutes; 94°C for 45s, 62°C for 45s, 72°C for 1 min, 30 cycles; 72°C for 10 minutes. Mix 5 μL of the PCR amplification product with 1 μL of loading buffer, spo...
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