1. Collection and storage of samples
Try to use fresh tissue materials. If the collected samples are not used immediately for extraction experiments, please store them at -20°C or -70°C, and avoid repeated freezing and thawing during this period. When using samples that have been repeatedly freeze-thaw or have not been refrigerated for a long time for DNA extraction, the extracted DNA fragments may be incomplete or the yield may be low. For bacteria and yeast cells that are difficult to break the cell wall, try to collect the bacteria in the logarithmic growth phase. The automatic dna extractor can solve these problems. The automatic system does not require manual intervention to achieve the best results. ,
2. Sample processing method
Fragmentation (and homogenization) of the sample material can improve the efficiency of lysis. After the tissue is broken, it can fully contact with the lysis solution, which is beneficial to the lysis. Tissue materials from different sources, according to the differences in tissue composition, sample processing methods are also different. Generally speaking, the methods of sample fragmentation mainly include liquid nitrogen grinding, homogenization and proteinase K digestion.
(1) Bacteria and yeast
Bacteria need to add Lysozyme (Lysozyme) to break the cell; yeast cells need to add Lyticase (Lyticase) to break the cell wall.
(2) Animal tissue
Generally, it is necessary to add proteinase K to digest the tissue, without liquid nitrogen grinding and homogenization, and cut it as much as possible (such as the tail of a mouse, add proteinase K for digestion for 1 to 4 hours). Fully broken can reduce the cracking time, you can use a glass homogenizer.
(3) Plant tissue
Must be ground and broken with liquid nitrogen.
3. DNA elution
Under low-salt conditions, DNA can be easily eluted from the silica membrane. The eluent uses a low-salt buffer (10mmol/I, Tris. HCI, pH8.5). When the pH value of the eluent is between 7.0 and 8.5, the elution efficiency is maximum. If ddH2O is used for elution, please ensure that the pH value is within this range. Preheating the elution buffer at 65°C for elution will improve the elution efficiency.
4. DNA detection
The OD260/OD280 of the purified genomic DNA will generally be between 1.7 and 1.9. During electrophoresis detection, the electrophoresis band has a lot to do with the amount of sample and the concentration of the agarose gel. The purified genomic DNA is ideally A single band, but often due to the operation in the extraction process and other reasons, the electrophoretic display is not clear as a slice of bands, which is a normal phenomenon.
(1) After the DNA solution is diluted 20 to 30 times, measure the OD260/OD280 ratio to determine the DNA content and quality.
(2) Take 2~5μL and electrophoresis on 0.7% Agarose gel to detect the molecular size of DNA.
(3) Take 2μg DNA, digest it with 10 units (U) HindⅢ overnight, and test whether it can be completely digested by electrophoresis on 0.7% Agarose gel (for RFLP, DNA must be completely digested).
5. DNA storage and stability
DNA molecules can exist stably under alkaline conditions. The eluent used in the kit is a buffer with pH 8.5, which can stably preserve DNA molecules. The pH value of ddH2O used in the laboratory is often less than 7.0, and DNA is prone to degradation when stored for a long time. For long-term storage, it should be stored at -20°C or -80°C, and repeated freezing and thawing should be avoided.