Agarose gel electrophoresis is a kind of electrophoresis method using agar or agarose as supporting medium. For samples with large molecular weight, such as large molecular nucleic acids, viruses, etc., agarose gels with large pore size can generally be used for electrophoretic separation. What are the common problems of agarose gel electrophoresis?

Fuzzy DNA bands

DNA degradation: nuclease contamination should be avoided in agarose gel electrophoresis test

Electrophoresis buffer is old: after the use of electrophoresis buffer, the ionic strength of the ionic strength is degraded, the PH value rises, the buffering capacity is weakened, which affects electrophoresis experiments, and it is recommended that the buffer be replaced frequently.

Inappropriate electrophoresis conditions: electrophoresis voltage should not exceed 20V/cm, temperature <30℃; huge DNA strand electrophoresis, the temperature should be <15℃; check whether all electrophoresis buffers have enough buffering capacity

Excessive amount of DNA uplift: DNA uplift in the gel should be reduced.

Excessive salt in DNA sample: remove excessive salt by ethanol precipitation before electrophoresis.

There is protein contamination: remove the protein by phenol extraction before electrophoresis

Irregular DNA bands migrate

For λ/Hind III fragment cosite complexation: heat the DNA at 65°C for 5 min before electrophoresis, then cool it on ice for 5 min.

Inappropriate electrophoresis conditions: electrophoresis voltage not more than 20V/cm; temperature <30°C; change electrophoresis buffer frequently.

DNA denaturation: dilute DNA with 20mM NaCI Buffer; do not heat before electrophoresis.

Weak or no DNA band

Insufficient sample volume of DNA: increase the sample volume of DNA; polyacrylamide gel electrophoresis is slightly more sensitive than agarose gel electrophoresis, and the sample volume can be appropriately reduced.

DNA degradation: avoid nuclease contamination of DAN

DNA out of the gel: shorten the electrophoresis time, reduce the voltage, and enhance the gel concentration.

For EB-stained DNA, the light source used is inappropriate: apply a short-wavelength (254 mm) UV light source

DNA bands missing

Small DNA bands coming out of the gel: shorten the electrophoresis time, reduce the voltage, and enhance the gel concentration.

DNA bands of similar molecular size not easily distinguishable: increase electrophoresis time, approve correct gel concentration.

DNA denaturation: do not heat NDA strands at high temperature before electrophoresis, dilute DNA with 20 mM NacI Buffer

DNA strands are huge and regular gel electrophoresis is not appropriate: analyze on pulse gel electrophoresis.

In summary, agarose gel electrophoresis experiments need to pay attention to the following points:

1. The most critical reagents when the system is prepared must make sure that they are still valid, or have not been contaminated: primers, enzymes, ultrapure water. It is best to put these things away by yourself, write your name on it and put it away, and remember to put a rubber band on the icebox, so that your icebox won't fall apart and the reagents will not fall off because of someone else's turning over the refrigerator. Otherwise, your reagents are contaminated, when the negative control wildly out of the band, and then go to find the source of contamination is very troublesome. And when you configure the system, you should pay attention to keep the experimental area clean, beforehand, you can use a small spray bottle for alcohol spraying to fix the DNA in the air, and you should wear gloves the whole time to avoid contaminating the system.

2. pcr system to feel accurate, it is best to use people have done a lot of old system to start.

3. To get a nice electropherogram, you can pour the gel on at the beginning of the PCR (provided that your total PCR time is around 1.5 hours and you are still in the lab after 1.5 hours.) , let the gel cool so that the upwellings of the gel will be more regular and its own pore size inside the gel will be more regular. If you are going to run the gel the next day, always remember to store the finished PCR samples at 4°C. When you do it the next day, it is recommended to cool the gel for about 25 minutes.

4. It is recommended that you use a lance for agarose gel electrophoresis, not only is it fast, but the samples are added to the wells at the same rate. Of course, it is better to use the imported gun head, but not the domestic gun head.

5. Regardless of the frequency of the electrophoresis chamber, I recommend replacing the electrophoresis solution every time agarose gel electrophoresis is performed to avoid the appearance of "^"-shaped bands.