RhD-T163P mutant and detection thereof
A rhd-t163p, 1.rhd-t163p technology, applied in the field of molecular biology, can solve the problems of inability to obtain correct results and difficult to determine the results, and achieve the effect of wide scientific research application value, high sensitivity and high precision detection
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Embodiment 1
[0034] The preparation of embodiment 1 DNA template
[0035] Whole blood genomic DNA was extracted using purchased kits. Specific steps are as follows:
[0036] (1) Take a sterile 2.0mL centrifuge tube and add 1mL of cell lysate.
[0037] (2) Gently shake the whole blood sample anticoagulated with EDTA until it is thoroughly mixed; then pipette 500 μL of blood sample into the above-mentioned centrifuge tube containing the cell lysate, and gently pour the centrifuge tube 5-6 times to mix well.
[0038] (3) Incubate at room temperature for 10 minutes (during this period, invert the centrifuge tube 2-3 times to mix well).
[0039] (4) Centrifuge at room temperature for 5 minutes at 12000 rpm.
[0040] (5) Use a pipette to slowly remove the supernatant as much as possible, and be careful not to suck out the white substance at the junction of the two phases.
[0041] (6) Vigorously mix with a vortex shaker (Votex) until the leukocytes are resuspended (10-15 seconds).
[0042] ...
Embodiment 2
[0052] Example 2 RHD487A>C allele detection:
[0053] Instruments: Veriti 96 PCR instrument, BIO-RAD Gel Doc XR+ gel imager (Bio-Rad, USA), gel electrophoresis (Beijing Liuyi Company).
[0054] Reagents: QIAamp DNA extraction kit (Qiagen, Germany); DNA Isolation Kit extraction kit (PELFREEZ, Beijing); PCR buffer, dNTP, Taq enzyme (ABI, USA); primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.
[0055] (1) RHD802A>G allele amplification: total reaction volume: 50 μL, containing 10 μL of PCR 5× buffer, 5.0 μL of DNA template, 1.0 μL of Taq polymerase (1U / μL), MgCl 2 The final concentration is 2.0mmol / L, the final concentration of dNTP is 200nmol / L, and the final concentration of specific upper and lower primers is 200nmol / L, and sterilized double-distilled water is added to a total volume of 50μL.
[0056] Reaction conditions: pre-denaturation at 95°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, followed by annealing at 61°C for 40 seconds, an...
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