Detection of apoptosis in tumor cells by DNA degradation and fracture assay
Detection of apoptosis in tumor cells by DNA degradation and fracture assay
DNA degradation fracture assay for detection of apoptosis in tumor cells can be used to (1) detect apoptosis in tumor cells and (2) detect the effect of drug treatment of tumors.
Operation method
Cellular DNA extraction
Principle
Apoptotic DNA degradation selectively occurs in inter-nucleosomal DNA, and electrophoresis after extraction of total cellular DNA or selective extraction of small molecular weight DNA allows examination of unique inter-nucleosomal fragment DNA.
Materials and Instruments
Tumor Cells Move I. Phenol extraction method: first use egg enzyme K, SDS to break the cells, digest protein, then use phenol and phenol-chlorine DNA size of 100-150kb. Second, formamide depolymerization method: broken cells as above, and then use a high concentration of formamide to depolymerize the combination of protein and DNA, and then dialysis to obtain DNA can be obtained DNA200kb or so. Third, glass rod winding method: use guanidine hydrochloride to lysate the cells, spread the lysate on ethanol, and then use a hook or U-shaped glass rod in the interface of the light stirring, DNA precipitation solution around the glass rod. Generate DNA about 80kb. Fourth, isopropanol precipitation method: basically the same as 1 method, only with two times the volume of isopropanol instead of ethanol, can remove small molecules of RNA (soluble state in isopropanol) V. Surfactant rapid preparation: use Triton X-100A or NP40 surfactant to break the cells, then use proteinase K or phenol to remove protein, ethanol precipitation or dialysis. Sixth, heating method rapid preparation: heating 96 ℃ -100 ℃, five minutes, then centrifuged and take the supernatant, can be used for PCR reaction. Seven, alkali denaturation rapid preparation: first with NaOH for 20 minutes, then add HCI neutralization, centrifugation and take the supernatant, containing a small amount of DNA. Caveat Factors affecting the mobility of DNA in agarose gel electrophoresis: ① DNA molecule size, the larger the DNA molecule, the slower the migration speed. ② Mobility is inversely proportional to agarose concentration. ③ DNA conformation: a. The same molecular weight of superhelical cyclic (type Ⅰ), incised cyclic (type Ⅱ), and linear (type Ⅲ) DNA, pass through the gel at different rates. b. Generally, the mobility type Ⅰ> type Ⅲ> type Ⅱ. c. The mobility of DNA is related to the size of DNA molecules. d. The mobility of DNA is related to the size of DNA molecules. ④ Voltage: the mobility is proportional to the applied voltage, but the effective separation range decreases when the voltage is too large. ⑤ Direction of electric field: the rate is equal in a single direction, changing the direction, the migration of very large molecules of DNA is slower. Separation of very large DNA by pulsed electric field gel electrophoresis. Common Problems Usually electrophoresed with a standard DNA fragment of known molecular weight, the size of the DNA specimen is known by comparison. If the bands are trailing, too much DNA has been added or the gel has not been prepared properly. If the molecular weight of the DNA is small or a piece is blurred, it indicates degradation of the DNA. For more product details, please visit Aladdin Scientific website.
PBS Lysis buffer Phenol Chloroform Isoamyl alcohol Ethanol Sodium chloride TE buffer TAE TBE Agarose gel electrophoresis Phosphoric acid-citric acid buffer Proteinase K R Nase enzyme Cell digest EDTA
Centrifuge UV spectrophotometer Electrophoresis apparatus Centrifuge tube Beaker Glass rod Measuring cylinder