Agarose gel electrophoresis of DNA

Summary

DNA electrophoresis can be used to (1) separate DNA fragments of different sizes, (2) identify target DNA fragments, and (3) purify and recover DNA fragments.

Operation method

electrophoresis

Principle

The basic principle of DNA agarose gel electrophoresis is to utilize the charge effect and molecular sieve effect that DNA molecules have when swimming in agarose gel. The charge effect refers to the fact that DNA molecules are negatively charged in a pH solution above the isoelectric point and move toward the positive pole in an electric field, and that the same amount of double-stranded DNA has almost equal net charge and can move at the same rate. Typically, the rate of DNA swimming decreases with increasing length of the DNA fragments and is proportional to the strength of the electric field. The molecular sieve effect refers to the fact that agarose, as a support medium, has a network structure that allows large molecules to receive greater resistance as they pass through, separating DNA fragments of different sizes accordingly. Generally, DNA agarose gel electrophoresis can separate DNA fragments of 50bp to millions of bp in length, and agarose gels of different concentrations and configurations are prepared according to actual needs.

Materials and Instruments

DNA fragments
Agarose Electrophoresis buffer (1XTAE or 0.5XTBE) Loading buffer Fluorescent insert dye (ethidium bromide or SYBR Gold) DNA size standard (DNA Marker)
Agarose gel electrophoresis instrument UV transilluminator or UV gel imager Microwave oven Pipettes Water baths

Move

1. tape off ends of washed and dried glue-making boards and place horizontally on workbench;
2. adjust the height of the comb;
3. weigh 0.24 g of agarose in 30 ml of 0.5 × TBE, completely dissolve the agarose particles in a microwave oven, and pour into the gelatinizing plate when it cools down to 45-50 篊;
4. after the gel has solidified, carefully remove the comb and tear off the tape;
5. mix the electrophoresis sample with bromophenol blue and spot the sample sequentially into the spiked wells; pUC185 _祃 +ddH2O3祃+bromophenol blue2祃共10祃 in 0.5 ml tube after mixing and spotting;
6. put the glue plate into the electrophoresis tank, add electrophoresis liquid, turn on the electrophoresis instrument, so that the nucleic acid samples to the positive electrode swimming;
7. cut off the power supply after electrophoresis, take out the gel, put it into 0.5礸/ml ethidium bromide (EB) solution for staining for 10-15min, rinse it with water and put it on the ultraviolet transmittance meter to observe the electrophoresis results and take photos to record.

Caveat

1. Factors affecting the rate of DNA migration in agarose gels：

（1） DNA molecular size and migration rate U are inversely proportional to logN (N is the number of base pairs). Molecules are equal in size and essentially equal in charge (DNA structural repetitiveness). The larger the molecule, the slower the migration. Equal amounts of spatially tightly structured electrophoresis is fast (superhelix > linear DNA)(2) Gel concentration, U is mobility, U0t Agarose concentration: logU=logU0 Kr is gel concentration, Kr is medium blocking factor. Different gel concentrations discriminate between different ranges of DNAt is the free electrophoretic mobility of DNA, the(3) DNA conformation: general migration rate superhelical loop > linear DNA > single-stranded open loop. When the conditions change, the situation will be reversed, also related to the concentration of agarose, current strength, ionic strength and EB content.(4) Applied voltage: At low voltage, the migration rate of linear DNA fragments is proportional to the applied voltage. The voltage applied to the gel should not exceed 5 V/cm to achieve good resolution.(5) Base composition and temperature: generally have little effect 4 -30 ℃.(6) Presence of embedded dye: reduce linear DNA mobility, (not advocated to be added in electrophoresis solution)(7) electrophoresis buffer (0.5 × TBE) composition and its ionic strength affects the mobility of DNA, no ions exist, nucleic acids basically do not swim, ionic strength is too large heat production, melting gel and lead to DNA denaturation, generally use 1 × TAE, 1 × TBE, 1 × TPE (all contain EDTA pH8.0).

2. Ethidium bromide (EB) is a carcinogenic agent, gloves should be worn during operation to minimize contamination of the countertop.

3. Electrophoresis indicator: There are two commonly used indicators for nucleic acid electrophoresis, bromophenol blue (Bb) is blue-violet; xylene cyanol (Xc) is blue, which carries less charge than bromophenol blue, and has a slower mobility than bromophenol blue in the gel.

Common Problems

Common Problems Cause Analysis

1. Run out of the DNA band blurred?

(1) DNA degradation: avoid nuclease contamination.

(2) Electrophoresis buffer is not fresh: after the electrophoresis buffer is used for many times, the ionic strength decreases, the pH value rises, and the buffering capacity weakens, thus affecting the electrophoresis effect. It is recommended to replace the electrophoresis buffer frequently.

(3) Inappropriate electrophoresis conditions: the electrophoresis voltage should not exceed 20V/cm and the temperature should be <30℃; for giant DNA strand electrophoresis, the temperature should be <15℃; check whether the electrophoresis buffer used has sufficient buffering capacity.

(4) Excessive amount of DNA samples: Reduce the amount of DNA samples in the gel.

(5) DNA sample contains too much salt: remove excessive salt by ethanol precipitation before electrophoresis.

(6) Protein contamination: remove protein by phenol extraction before electrophoresis.

(7) DNA denaturation: do not heat the sample before electrophoresis and dilute the DNA with 20 mM NaCl buffer.

2. There is irregular DNA band migration?

(1) For λ/Hind III fragment cos site recombination: heat DNA at 65℃ for 5 minutes before electrophoresis, then cool on ice for 5 minutes.

(2) Inappropriate electrophoresis conditions: electrophoresis voltage should not exceed 20V/cm, temperature <30℃; change the electrophoresis buffer frequently.

(3) DNA denaturation: do not heat before electrophoresis, dilute DNA with 20mM NaCl buffer.

3. run out of DNA bands weak or no bands?

(1) The amount of DNA sample is not enough: increase the amount of DNA sample.

(2) DNA degradation: avoid DNA nuclease contamination.

(3) DNA out of the gel: connect the electrodes correctly to avoid inserting the opposite, shorten the electrophoresis time, reduce the voltage, and enhance the gel concentration.

(4) Inappropriate light source: For DNA stained with EB, use a short wavelength (254nm) ultraviolet light source.

4. run out of DNA bands missing incomplete?

(1) If it is a small DNA band, it may run out of the gel, you can shorten the electrophoresis time or reduce the voltage or enhance the gel concentration.

(2) DNA bands of similar molecular size are not easy to distinguish, electrophoresis time should be extended and the correct gel concentration should be approved.

(3) DNA denaturation: do not heat before electrophoresis, and dilute the DNA with 20 mM NaCl buffer.

Experimental Methods Source Source of Molecular Cloning Lab Guide

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