The objectives of the experiment are to understand and master the methods of detecting the quality of DNA as well as the methods of DNA quantification; to understand the factors affecting the rate of DNA swimming in agarose gel; to train the operation of agarose gel electrophoresis of DNA and restriction endonuclease operation of DNA. Source: Laboratory Course in Genetics
Operation method
zymography
Principle
At pH 8.0~8.3, nucleic acid molecule bases are almost not dissociated, phosphoric acid is all dissociated, and nucleic acid molecules are negatively charged and move toward the positive pole during electrophoresis. The use of appropriate concentration of gel medium as electrophoresis support, under the action of molecular sieve, so that the nucleic acid molecules with different molecular size and conformation of the swimming rate of a large difference, so as to achieve the separation of nucleic acid fragments to detect the size of the purpose. After the nucleic acid molecules are embedded with fluorescent dyes (e.g., EB), the location of the nucleic acid fragments can be observed under UV light.
Materials and Instruments
Rice DNA BAC cloned DNA Move 1. 10 μl of DNA was taken on a 0.8% gel assay; Caveat 1. Enzymatic digestion does not have to be overnight, but the longer the reaction time the less enzyme is used. Compared with the enzymatic digestion of vector/insertion fragments, the concentration of enzymes used for genomic DNA digestion is higher.Compared with the enzymatic digestion of identification vector/insert fragment, the concentration of enzyme used for the enzymatic digestion of genomic DNA is higher, generally 5U of endonuclease should be added for 1μg of genomic DNA. 2. If the electrophoretic band is much brighter near one end of the sample well than the other, it indicates that the digestion is incomplete. This may be due to: ① the reaction time is not long enough (if the reaction overnight can be excluded); ② the amount of enzyme is not enough or the enzyme is partially inactivated; ③ if the initial DNA samples are dissolved in TE buffer and in the enzyme reaction in a larger volume, the TE buffer may inhibit the enzyme reaction of the EDTA, then you can use ethanol to reprecipitate the DNA, then dissolved in a small amount of TE buffer or SWD. 3. The relative molecular mass of the selected DNA should cover a wide range of relative molecular mass, such as λHind III can cover the range of 500bp to 23kb. 4.The brightness of the bands in each lane of the digested DNA sample should be the same, otherwise, it indicates that the concentration of each sample is not consistent.The concentration of DNA is usually difficult to be determined accurately, so the amount of DNA can be adjusted by adjusting the volume of the sample. For more product details, please visit Aladdin Scientific website.
Agarose Restriction endonuclease DraI EcoRI EcoRV HindIII Gel
Water bath agarose electrophoresis apparatus
2. The DNA was adjusted to a concentration of 300-400 ng/ 2. Adjust the concentration of DNA to 300-400 ng/μl. ;
3. Carefully read the product instructions for any of the enzymes that will be used to familiarize yourself with the reaction conditions and the storage concentrations for the enzyme cleavage (10U-50 U/). μl );
4. Calculate the exact amount of each reagent needed for the reaction conditions: (in a 0.5 ml tube)
DNA (3-5 mM) 10 μl
buffer reaction ´10 1.5 μl
Enzyme (15 U/) Enzyme (15 U/) ) 0.8 μl Enzyme (15 U/ μl ) 0.8 μl (on ice)
ddH2O 2.7 μl
Mix well and centrifuge briefly;
5. 37 ℃ warm bath for 1-2 hrs (pure DNA) or 10 hrs (crude DNA);
6. Add sample buffer to terminate the digestion reaction, or heat at 65 ℃ for 10 min to inactivate the enzyme;
7. Electrophoresis to detect the efficiency of the enzyme digestion:
1/10 of the amount of each sample was taken to detect it by agarose electrophoresis, and the method of gel preparation and spotting was the same as above.