The PCR method detects myosomes

PCR is an in-body method established in the mid-1980DNA amplification technology, the basic principle is enzymatic DNA synthesis reaction, that is, in the presence of DNA templates, citations and DNA, through the role of DNA polymerase, so that the DNA chain amplification extension. The method has the characteristics of high sensitivity, strong specificity and fast, but its requirements for experimental environment are strict, the experimental cost is high, and sometimes there is false positive phenomenon.

1, < a href ""> instrument equipment

ultra-clean workbench, PCR meter, electrophoresis instrument, gel imaging analysis system, desktop centrifuge, vortex mixer, etc.

2, experiment "> reagents

selected the U.S. Stratagene company produced by the branched medical test kit (with lead, positive control, internal control, StrataClean resin, buffer), dNTP, TapDNA polymerase, buffer,agar sugar, mineral oil.

3, experimental operation

PCR reaction should be carried out in a sterile environment.

(1) sample collection: the cells to be tested with no double resistanceculture base culture 7d, with sterile containers to take liquid 500ul, 4 degrees C to be stored for test.

(2) template production: in sterile conditions, takecell culture on clear 1 00ul in a sterile 0.5 ml plastic centrifuge tube, covered with a lid, 95 degrees C water bathheating 5min.

(3) Open the lid, add StrataClean resin10ul to the tube, cover the lid, vortex suspension mix, centrifuge 5-10s, absorb the upper clear to a new plastic centrifuge tube, the template is finished, 4 degrees C save.

(4) PCR reaction: the most suitable conditions for the reaction system are: 10mmol/lTris-HCL (pH8.38); 50mmol/lHCL; 1.5-2.5mmol/lMgCL2; 200umol/LDNTP; 2UDDNAap polymerase. The total reaction system is 50ul, and the reaction water with deionized water needs to be irradiated with 12000uw/cm2 UV lamp. The reaction is as follows table:

table reaction program

(2) adds the following ingredients in turn: 0.4uldNTPs (25mmol/l), 0.4ulTaqDNA polymerase (5U/ul), 2ul citations.

(3) plus 2ul deionized water, total volume 45ul.

(4) plus 2ul templates made into the reaction system.

(5) positive control, the internal control of each 5ul added to their respective reaction system.

(6) takes a centrifugal tube containing the above reaction system and adds 5ul deionized water as a negative control tube.

(7) adds 100ul mineral oil to the reaction system.

(5) agarose gel electrophoresis: after the PCR reaction, agarose gel electrophoresis, agarose gel concentration 2%. After electrophoresis, gel imaging analysis results.

(6) results analysis: This method is a qualitative method for detecting myosomes, in electrophoresis lanes, MARKER, positive control, internal control will appear different electrophoresis strips, when the sample swimming lanes appear bright strips, and the position between the positive control and negative control strip position, the sample can be considered to be contaminated by myosome. Sometimes more than one lane is found, possibly due to the sample infecting more than two myons. If the strip in the lane is looming, the sample can be redoed if there is suspected mycosm contamination.