After running for so many years of electrophoresis, do you know its principle? Many people definitely don’t know, because it’s too basic. Even when I first entered the laboratory, I didn’t ask about the principle of agarose gel electrophoresis, and the brother only talked about the negative charge of DNA and it will run to the positive electrode; EB is embedded in In the DNA strand, it can be seen by ultraviolet radiation. I don't understand the rest.

Principles of Gel Electrophoresis

DNA molecules in conventional electrophoresis buffer (pH solution higher than the isoelectric point), the hydrogen ion of 5'phosphate is neutralized due to dissociation, leaving only phosphate ion, so it is negatively charged, and therefore moves to the positive electrode in the electric field . Also, because agarose gel has a molecular sieve effect, under a certain electric field strength, due to the charged properties of various molecules in the sample to be separated and the differences in the size and shape of the molecules themselves, different DNA molecules have different migration speeds. Therefore, agarose gel electrophoresis can not only separate DNA with different molecular weights, but also separate DNA molecules with the same molecular weight but different configurations, such as the three configurations of plasmids. Let's talk about TAE and TBE.

TAE

TAE is Tris-acetic acid, the most widely used and most commonly used buffer. Its characteristic is that the supercoil electrophoresis is more in line with the actual relative molecular mass, and the migration rate of double-stranded linear DNA is about 10% faster than other buffers. When the DNA fragment is larger than 13kb, the TAE buffer will achieve better Separation effect, in addition, it is best to use TAE buffer for electrophoresis when recovering DNA fragments. The disadvantage of TAE is that the buffer capacity is small, and the long-term electrophoresis heats up, unless there is a circulation device to exchange the two-pole buffer.

Application: Separation of large fragment nucleic acid molecules, recovery of DNA by agarose gel electrophoresis, restriction enzyme detection, PCR reaction.

50x TAE Buffer (Tris-Acetate-EDTA) configuration is as follows

242 g ----------- Tris base

57.1 ml ----------- Acetic acid

100ml ------------ 0.5M EDTA

Add ddH2O to 1 liter and adjust pH to 8.5

TBE

TBE is Tris-boric acid, with large buffer capacity, but low solubility, not easy to store for a long time, and easy to produce precipitation. It has a good separation effect on fragments smaller than 1kb, but the recovery rate of DNA fragments is reduced due to the interaction with agarose to generate non-covalently bound tetrahydroxyborate complexes, and the borate ions may affect the follow-up Enzymatic reaction, therefore, TBE electrophoresis is generally not recommended for experiments that require gel-cutting and recovery.

This buffer is commonly used for general electrophoresis that only does detection, such as detection after DNA, plasmid, and RNA extraction. Because the resolution of the glue that TBE runs out will be higher.

It is often formulated into a 10× mother liquor, which can be placed for a long time without forming a precipitate, and is diluted to 0.5× when used.

10x TBE Buffer (Tris-Borate-EDTA) configuration is as follows

108 g--------------- Tris base

55 g --------------- Boric acid

9.3 g --------------- Na4EDTA

Add ddH2O to 1 liter.

The pH is 8.3 and requires no adjustment.

RNA formaldehyde denaturing glue

After extracting the total RNA of the sample, the quality of the RNA is generally judged according to the gel electrophoresis of the RNA. Since RNA is easy to form a secondary structure, formaldehyde denaturing gel is often used for RNA electrophoresis, and the obtained electrophoresis pattern can truly reflect the quality of RNA.

5X formaldehyde gel electrophoresis buffer

0.1mol/L MOPS (PH7.0)

40mmol/L sodium acetate

5mmol/L EDTA(ph8.0)

Before electrophoresis, heat the RNA at 65°C for 5 min, and quench it on ice to eliminate the secondary structure of RNA. It is recommended to add 0.5-1.0 ul ethidium bromide (EB, concentration 1.0 mg/mL) to the RNA sample before loading, instead of adding EB to the gel, so that the background after electrophoresis is low.