Mitochondrial DNA (mtDNA) is an important material for molecular evolution research and maternal genetic research, but the traditional two-step mtDNA extraction method is to gently lyse cells, isolate mitochondria, and then extract mtDNA from mitochondria. The conditions for gentle lysis of cells in this method are very difficult to control and need to be optimized separately for different tissue materials. This method overcomes the above disadvantages and has the following advantages:
1. There is no need to purify mitochondria separately, the column method purification, the operation is simple and fast.
2. The obtained mtDNA can be directly used for PCR and digestion.
3. Every 107 animal cells can get about 100ng of mtDNA, and every gram of tissue can get 3-5 ug mtDNA.
4. Note: This product is only suitable for animal materials, not for plant materials, because plant mitochondrial DNA is generally very large and very difficult to mention.
50 times packaging
Solution A13 mL
Solution B13 mL
Solution C18 mL
50 sets of centrifugal adsorption columns
Universal column wash 50 mL
DNA eluent 10 mL
1 manual
It can be stored at room temperature for short-term transportation at room temperature; it is best to store at 4 ℃ for long-term storage.
no
1. Pretreatment of cultured cells
1. Treat about 107 cultured cells with 0.25% pancreatin digestion solution prepared by yourself, centrifuge to collect and discard the supernatant.
2. Wash the cells twice with buffers such as PBS or TBS. The cell pellet obtained by centrifugation is directly used for mtDNA extraction.
Two: tissue cell pretreatment
3. Use scissors to shred 1g of fresh animal tissue (the best size of sesame) and transfer it to a 1.5mL plastic centrifuge tube for mtDNA extraction. Note: It is best not to use frozen animal tissue, because the ice crystals formed during the freezing process will pierce the mitochondria, release their DNA and be degraded by DNase in the cytoplasm, which affects the recovery rate.
Three: blood pretreatment
4. Leave 3-5 mL of anticoagulated peripheral blood for 30 minutes or centrifuge at low speed for 5 minutes to take the leukocyte layer.
5. Wash the white blood cells twice with self-prepared PBS. The obtained white blood cell pellet is used for mtDNA extraction.
Four: mtDNA extraction
5. Add 250 uL of ice bath solution A to the cell pellet or shredded tissue, and thoroughly disperse by pipetting. If it is shredded tissue, do not wait for the tissue to dissolve before proceeding to the next step.
6. Add 250 uL of normal temperature solution B (if solution B is precipitated, it must be heated and dissolved at 37 ° C before use and cooled to room temperature before use), gently invert and mix repeatedly 10 times. Never shake violently.
7. Leave on ice for 6-8 minutes. Be careful not to exceed 8 minutes.
8. Add 350 uL of ice bath solution C, mix gently for 4-6 times, white precipitate can be seen, and then put on ice for 20-25 minutes.
9. Centrifuge at 12000-15000 g at room temperature for 10 minutes, and carefully transfer the supernatant to the centrifuge adsorption column. Due to the large specific gravity of the solution at this time, it is sometimes normal for the sediment to float, and it is sufficient to avoid the floating sediment when taking the supernatant.
10. Let it sit for 5 minutes to allow the DNA to fully bind to the spin column. This step is very important.
11. Centrifuge at 12000-15000 g at room temperature for 1 minute, and discard the waste solution in the collection tube.
12. Add 500 uL of universal column wash solution, centrifuge at room temperature 12000-15000 g for 1 minute, and discard the waste solution in the collection tube. Note: The general column washing solution contains ethanol. After use, the cap needs to be tightened and stored, otherwise the ethanol will evaporate.
13. Repeat the previous step once.
14. Centrifuge at 12000-15000 g at room temperature for 1 minute, and spin off the residual liquid. This step can not be omitted, otherwise the residual ethanol will affect the subsequent use of DNA (such as DNA can not be precipitated into the sample well when loading).
15. Place the centrifugal adsorption column in a new 1.5 mL plastic centrifuge tube, add 30-50 uL of pre-heated DNA eluent at 65-80 ° C, and leave at room temperature for 2 minutes. Normal temperature DNA eluent can also be used for elution, but the yield is slightly reduced.
16. Centrifuge at 12000-15000 g at room temperature for 1 minute, and the bottom solution of the centrifuge tube is mtDNA.
17. Because our company's centrifugal adsorption column has a strong ability to bind DNA, if you add 30-50 uL of DNA eluent to the centrifugal adsorption column, it can often elute a lot of mtDNA (equivalent to the first elution 30%), but be careful not to use the mtDNA solution obtained in the previous step for elution.
18. Centrifuge at 12000-15000 g for 1 minute. The solution at the bottom of the centrifuge tube is the mitochondrial DNA. Every 107 animal cells can usually get about 100ng of mtDNA, and every gram of tissue can usually get 3-5 ug of mtDNA. If DNA needs to be concentrated, we can use our nucleic acid concentrate.
19. Due to the mechanical breakage of DNA, DNA may not show a specific band during electrophoresis, but this mtDNA solution can be used directly for PCR.
Mitochondria
Mitochondria are organelles that synthesize ATP and fatty acids. Each cell has multiple mitochondria, and each mitochondria has multiple mitochondrial genomes. For example, there are more than 20 mitochondria in each S. cerevisiae cell and several human lymphocytes. There are 500-2500 liver cells and thousands of oocytes. Except for a few lower eukaryotes whose mitochondrial genomes are linear DNA, the mitochondrial genomes of other organisms are all circular DNA. The mitochondrial genome lacks histones, so there are no nucleosomes. Mammalian mitochondrial DNA has no introns. Almost every pair of nucleotides is involved in the composition of a gene, and the sequences of many genes overlap. The mitochondrial genomes of different species vary greatly in size. The trend in animals is that the higher the species, the smaller the mtDNA. Common species mtDNA size list:
Species mtDNA size (KB) example
Vertebrate 15.7-17.5 mice are 16,295bp;
16,569bp for humans
Insects 14.5-17.9
Protozoa 18.5-40.0
Fungus 18.9-95.0 yeast is 75Kb, Aspergillus is 32Kb
Algae 15.0-18.0
Higher plant 120-2700 maize mtDNA is 600 Kb
Human mtDNA only has a D-loop region (D-loop, involved in replication initiation) and another 87 bp are not coding genes, the remaining sequences encode a total of 13 proteins (cytochrome b, 3 subunits of cytochrome oxidase, ATPase 2 subunits and 7 subunits of NADH dehydrogenase), 24 rRNA (including 1 16SrRNA, 1 12SrRNA and 22 tRNA). Human neuromuscular degeneration diseases such as Leber's hereditary optic neuropathy, Parkinson's disease, Alzheimer's disease, mitochondrial encephalomyopathy, maternal diabetes and deafness are all related to mitochondrial genes. Compared with nuclear DNA, mtDNA has the following characteristics: â‘ The mutation rate is high, which is about 10-100 times that of nuclear DNA, which is helpful to detect the changes of genes in a short period of time, and to compare the same genes of different species The difference between these species determines the evolutionary genetic relationship; â‘¡ maternal inheritance, this is because the animal sperm has very little cytoplasm, and the offspring mtDNA is basically derived from egg cells, so individuals with the same mtDNA sequence must From a common female ancestor.
Strapping machines use strapping straps to strap products or packages, and then tighten and combine the two ends by hot-melt hot-melt bonding. The function of the packing machine is to reinforce the packaged items, so that the items will not be scattered due to the loose binding during the transportation and storage, and they should also be bundled neatly and beautifully.
Automatic Strapping Machine,Auto Strapping Machine,Small Strapping Machine,Fully Automatic Strapping Machine
Jining Myway Machinery Co., Ltd. , https://www.mwpackall.com