The preparation of genomic DNA is an important step in the study of gene structure and function, and it is usually required that the length of the obtained fragment is not less than 100-200 kb. In the DNA extraction process, various factors that cause DNA fragmentation and degradation should be avoided as much as possible to ensure the integrity of the DNA and lay the foundation for subsequent experiments. Generally, eukaryotic genomic DNA is 107-9 bp, which can be extracted from fresh tissue, cultured cells or cryopreserved tissue cells. It is often digested with proteinase K in the presence of reagents such as EDTA and SDS, followed by phenol extraction. Realized. The DNA obtained by this method can be used not only for enzyme digestion but also for Southern analysis, and can also be used for PCR template, library construction and the like.

Different material processing methods are used depending on the source of the material, and the subsequent DNA extraction methods are generally similar.

But both should consider the following two principles:

(1) Preventing and inhibiting the degradation of DNA by DNase;

(2) Minimize mechanical shear damage to DNA in solution.

First, reagent preparation

1. TE: 10 mM Tris-HCl (pH 7.8); 1 mM EDTA (pH 8.0).

2. TBS: 25 mM Tris-HCl (pH 7.4); 200 mM NaCl; 5 mM KCl.

3. Lysis buffer: 250 mM SDS; Proteinase K was added to 100 mg/ml before use.

4.20% SDS

5.2mg/ml proteinase K

6.Tris saturated phenol (pH 8.0), phenol/chloroform (phenol: chloroform = 1:1), chloroform

7. Anhydrous ethanol, 75% ethanol

Second, the operation steps

(1) Material handling

1. Fresh or frozen tissue treatment:

(1) Take the tissue block 0.3-0.5cm3, cut it, add TE 0.5ml, and transfer it to the homogenizer for homogenization.

(2) Transfer the homogenate to a 1.5 ml centrifuge tube.

(3) Add 20% SDS 25 ml, proteinase K (2mg/ml) 25 ml, and mix.

(4) 60 ° C water bath 1-3hr.

2. Culture cell processing:

(1) After the cultured cells are suspended, they are washed once with TBS.

(2) Centrifuge 4000g × 5min, remove the supernatant.

(3) Add 10 volumes of lysis buffer.

(4) 50-55 ° C water bath 1-2hr.

(two) DNA extraction

1. Add an equal volume of saturated phenol to the above sample treatment solution, gently and thoroughly mix for 3 min.

2. Centrifuge 5000g x 10min and take the upper aqueous phase to another 1.5ml centrifuge tube.

3. Add an equal volume of saturated phenol, mix, centrifuge 5000g × 10min, take the upper water phase to another tube.

4. Add an equal volume of phenol/chloroform, mix gently, centrifuge 5000g×10min, and take the upper aqueous phase to another tube. If the water phase is still not clear, repeat this step several times.

5. Add an equal volume of chloroform, mix gently, centrifuge 5000g×10min, and take the upper water phase to another tube.

6. Add 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol and mix by inverting.

7. After the floc appears, centrifuge 5000g×5min and discard the supernatant.

8. The pellet was washed with 75% ethanol, centrifuged at 5000 g x 3 min, and the supernatant was discarded.

9. Evaporate the ethanol at room temperature. After the precipitate is nearly transparent, add 50-100 ml of TE to dissolve overnight.

(3) DNA quantification and electrophoresis detection

1. DNA quantification:

DNA has the largest absorption peak at 260 nm, the protein has the largest absorption peak at 280 nm, and the salt and small molecules are concentrated at 230 nm. Therefore, the DNA concentration can be measured by spectrometry at a wavelength of 260 nm, and the OD value of 1 corresponds to about 50 μg/ml of double-stranded DNA. For example, using a 1 cm optical path, dilute the DNA sample n times with H2O and use H2O as a blank control. Calculate the concentration before dilution of the sample according to the OD260 value read at this time: DNA (mg/ml) = 50 × OD260 reading × Dilution factor / 1000.

The pure OD260/OD280 of DNA is 1.8, so the purity of DNA can be estimated based on the value of OD260/OD280. If the ratio is higher, the RNA is contained, and a lower ratio indicates the presence of residual protein. The ratio of OD230/OD260 should be between 0.4 and 0.5. If the ratio is higher, there is residual salt.

2. Electrophoresis detection:

1 μg of genomic DNA was electrophoresed on a 0.8% agarose gel to determine the integrity of the DNA, or whether the concentrations of the multiple samples were the same. After the end of the electrophoresis, there should be a single high molecular weight band near the spotting well.

Third, matters needing attention

1. All supplies require high temperature and pressure to inactivate residual DNase.

2. All reagents were prepared in autoclaved double distilled water.

3. Operate with a large dropper or tip to minimize the possibility of breaking DNA.

4. The purity of the DNA extracted by the above method can satisfy the purpose of general experiments (such as Southern hybridization, PCR, etc.). DNA purification can be performed if required.

Extraction and purification of plasmid DNA by alkaline lysis

Bacterial plasmids are a class of double-stranded, closed-loop DNA ranging in size from 1 kb to over 200 kb. All kinds of plasmids are autonomously occurring genetic components that exist in the cytoplasm and are independent of the chromosomes of the cells. They are usually stably and stably in the extrachromosomal state, but under certain conditions, they are reversibly integrated into the host chromosome. It replicates as the chromosome replicates and is passed through the cell division to the offspring.

Plasmids have become the carrier molecules of the most commonly used gene clones at present, and it is important to obtain a large number of purified plasmid DNA molecules. There are many methods available for the extraction of plasmid DNA. In this experiment, plasmid DNA was extracted by alkaline lysis.

Alkaline lysis is one of the most widely used methods for preparing plasmid DNA. The basic principle is that when the bacteria are lysed in NaOH and SDS solution, the protein and DNA are denatured. When the neutralizing solution is added, the plasmid DNA molecule can It is rapidly renatured and is in a dissolved state. It remains in the supernatant during centrifugation; the protein and chromosomal DNA are invariant and flocculate, and can be precipitated during centrifugation.

The method of purifying plasmid DNA usually utilizes two properties of relatively small and covalently closed plasmid DNA. For example, cesium chloride-ethidium bromide gradient equilibrium centrifugation, ion exchange chromatography, gel filtration chromatography, polyethylene glycol fractionation precipitation, etc., but these methods are relatively expensive or time consuming. For small-prepared plasmid DNA, the residual protein and RNA are removed by simple steps such as phenol, chloroform extraction, RNase digestion and ethanol precipitation. The purified plasmid DNA can satisfy bacterial transformation, DNA fragment separation and digestion, and conventional Subcloning and probe labeling are required, so they are commonly used in molecular biology laboratories.

Air Quality Detector

How assured are you that you are breathing in healthy air? Have you ever thought about the impact unhealthy air can have on your health? Knowing that breathing is an every day, all day process which one cannot do without as long as they live, you might as well want to pay some attention to the quality of the air you inhale in your home, office or car.

Indoor Air Quality Monitor is a carefully designed air quality monitor. It is a highly portable device with excellent efficiency. Feel free to use this to your benefit everywhere you find yourself. It is suitable for use indoors. Again, it does not support Eve cloud and data sales but it does guarantee the privacy of all your data.For clean, healthy, and fresh air, this is just right for you and your loved ones. Unpleasant VOC levels which stem from our everyday furniture, toys, electronics, carpet, and many others are detected by this air quality monitor. Everyday activities like cooking and cleaning are closely monitored, showing how they impact the quality of air in your home or office. Plus it gives reliable recommendations on steps to be taken to improve your air quality.

The IQAir Air Monitor is one of our favorite premium products on this list. It does the same basic job as the cheapest air monitors, but the readings are generally more accurate, and they offer better connectivity. In simple terms, if you really want the most accurate reading, you should spend more money. But not everyone needs to know exactly what their home`s levels of CO2 are.

Air Quality Detector,Tvoc Monitor With Usb Charge,Formaldehyde Detector Monitor,Tvoc Gas Analyzer

Xi'an Lonn M&E Equipment Co., Ltd. , https://www.smartmeasurer.com