Tissue sample DNA purification
Most animal tissues can be efficiently lysed with lysis buffer and protease/proteinase K. The tissue needs to be cut into small pieces before adding the lysate. If possible, it is recommended to use the TissueLyser or mortar to pre-homogenize. Skeletal muscle, heart, skin and other tissues contain contractile proteins, connective tissue and collagen, so it is necessary to fully digest with protease/proteinase K.
Pre-cracking is required for FFPE samples for DNA purification. Formalin can be removed by washing with PBS, and paraffin can be removed by xylene and ethanol. Increasing the temperature of incubation after protease digestion helps reverse cross-linking to better release DNA from FFPE tissue, which helps to increase DNA yield and improve downstream detection performance. The DNA obtained from FFPE is generally smaller than the DNA obtained from fresh or cryopreserved samples; the extent of DNA degradation depends on the type of sample, storage time and fixed conditions.
Purify DNA from trace samples
The amount of DNA in a small sample is very low, usually less than 5 ng of DNA, and carrier RNA is usually required to increase yield (without affecting downstream assays). If carrier RNA is required, the concentration measurement will be biased due to the use of carrier RNA when measuring OD (because RNA is also absorbed at 260 nm).
DNA purification from a small sample using the QIAamp DNA Micro Kit is recommended. The kit uses a MinElute silica membrane column with an elution volume as low as 20 μl and a two-step wash to remove contaminants such as PCR inhibitors, maximally from microsamples. Purify the DNA.
Purify DNA from blood samples
The blood sample may be fresh or frozen whole blood or dried blood. Since the blood sample will rapidly coagulate after collection, EDTA, citrate or heparin is usually used for anticoagulation during blood collection. The anticoagulated blood sample can be directly used for DNA purification. It should be noted that the DNA purification method used needs to be able to remove the above anticoagulant to avoid interference with downstream PCR detection. Whole blood can also be subjected to DNA purification by performing leukocyte enrichment before DNA purification.
The yield of purified DNA from the blood depends largely on the number of white blood cells in the blood, and the number of white blood cells is strongly related to the donor's condition in the blood (eg anemia or infection can cause changes in the number of white blood cells). This must be considered before DNA purification. In addition, some animals have nuclear blood, which means that this type of sample contains more DNA. In the purification of DNA from such blood samples, the amount of sample loading needs to be reduced by about 10 times.
Purify DNA from cells
Mammalian cells can be lysed using proteinase K
Yeast cells need to first treat the cell wall with lysozyme and then digest it with protease or proteinase K.
Many bacterial cells can be lysed using lysis buffer and protease/proteinase K. Some bacteria, such as Gram-positive bacteria, require pretreatment with lysozyme to treat the cell wall.
Bacterial cells need to be pre-precipitated from biological fluids, and then the bacterial DNA is purified from it. The swab samples need to be pre-treated with fungicide and then centrifuged.
The use of mechanical lysis is better than digestion with enzymes, especially for Gram-positive bacteria or fungi.
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Thank you for your interest in Shanghai Jiapeng's products. In addition to this product introduction, the company also has three kinds of UV analysis, nucleic acid protein detector, gel imaging analysis system, photochemical reactor, constant current pump, automatic partial collector, etc. Introduction, if you are interested in Shanghai Jiapeng products, please contact us. Thank you!
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