PCR is an in vitro DNA amplification technology established in the mid-1980s. Its basic principle is enzymatic DNA synthesis, which is the role of DNA polymerase in the presence of DNA templates, primers and deoxyribonucleic acid. DNA strand amplification extends. The method has the characteristics of high sensitivity, high specificity and rapidity, but its requirements on the experimental environment are strict, the experiment cost is high, and sometimes there is a phenomenon of false positive. 1, equipment Ultra-clean workbench, PCR instrument, electrophoresis instrument, gel imaging analysis system, benchtop centrifuge, vortex suspension, etc. 2, experimental reagents The Mycoplasma detection kit (with primers, positive control, internal control, StrataClean resin, buffer), dNTP, TapDNA polymerase, buffer, agarose, mineral oil was prepared from Stratagene, USA. 3. Experimental operation The preliminary operation of the PCR reaction should be carried out in a sterile environment. (1) Sample collection: The cells to be tested were cultured for 7 days in a medium without a double-antibody, and 500 ul of the supernatant was taken in a sterile container and stored at 4 ° C for testing. (2) Preparation of template: Under sterile conditions, 100 μl of the cell culture supernatant was placed in a sterile 0.5 ml plastic centrifuge tube, the lid was capped, and heated in a 95 ° C water bath for 5 min. (3) Open the lid, add StrataClean resin 10ul to the tube, cover the lid, mix with the vortex suspension, centrifuge for 5-10s, and pipette the supernatant into a new plastic centrifuge tube. The template is finished and stored at 4 °C. (4) PCR reaction: The optimum conditions of the reaction system are: 10 mmol/l Tris-HCL (pH 8.38); 50 mmol/l HCL; 1.5-2.5 mmol/l MgCL2; 200 umol/ldNTP; 2 UDap DNA polymerase. The total reaction system is 50 ul, and the deionized water for the reaction is irradiated with a 12000 uw/cm2 ultraviolet lamp. The response is as follows: Reaction program 2 The following ingredients were added in sequence: 0.4 nt NTPs (25 mmol/l), 0.4 ul of Taq DNA polymerase (5 U/ul), 2 ul of primers. 3 plus 2ul deionized water, total volume 45ul. 4 plus 2 ul of the prepared template into the reaction system. 5 positive control, 5 ul of each internal control was added to the respective reaction system. 6 Take a centrifuge tube containing the above reaction system, and add 5 ul of deionized water as a negative control tube. 7 Add 100 ul of mineral oil to the reaction system. (5) Agarose gel electrophoresis: After the end of the PCR reaction, agarose gel electrophoresis was carried out, and the agarose gel concentration was 2%. After the end of the electrophoresis, the gel was imaged for analysis. (6) Analysis of results: This method is a qualitative method for detecting mycoplasma. In the electrophoresis lane, different electrophoresis bands appear in MARKER, positive control and internal control. When the test sample shows bright bands, the position is positive. Between the control and negative control strip positions, the sample is considered to be contaminated with mycoplasma. Sometimes there will be more than one lane, which may be caused by the infection of more than two types of mycoplasma. If the band in the lane is looming, suspected mycoplasma contamination and redo the sample. Pollock Fillet,Petite Hake Fillets,Filleting Hake,Cape Hake Fillets ZHEJIANG EVERNEW SEAFOOD CO.,LTD , https://www.evernewseafood.com Program Circulation Temperature / °C Time/min 94 2 1 1 50 2 72 2 94 1 2 40 50 1 72 2
1 Add 35.2 ul of deionized water and 5 ul * 10 Taq reaction buffer to a 0.5 ml plastic centrifuge tube.