Tris acetate gel experimental steps
Experimental preparation
1. ** Reagents and materials **: prepare Tris, glacial acetic acid, ethylenediaminetetraacetic acid (EDTA), agarose, deionized water, samples (substances to be tested), nucleic acid molecular weight standards, electrophoretic buffers, ethidium bromide (EB) or other nucleic acid dyes, pipettes and gun heads, glue moulds, combs, electrophoretic tanks, power supplies, etc.
2. ** Solution preparation **:
- ** 50 × TAE buffer (Tris-acetic acid-EDTA buffer) **: Weigh 242g Tris base, add 57.1ml glacial acetic acid, 100ml 0.5M EDTA (pH 8.0), bandwidth evaluation with deionized water to 1L. Dilute 50 times to 1 × TAE working buffer when using.
- ** agarose gel **: Weigh an appropriate amount of agarose according to experimental needs. For example, a 1% - 2% agarose gel is commonly used for DNA detection, that is, if 100ml of gel is formulated, 1% of the gel is weighed by 1g agarose, and 2% of the gel is weighed by 2g agarose. Add the agarose to an appropriate amount of 1 × TAE working buffer, heat and stir until completely dissolved.

Glue making
1. ** Assemble the glue making mold **: Wash and dry the glue making mold, place it on a water platform, insert a comb, and ensure that the bottom of the comb is kept at an appropriate distance from the bottom of the mold (generally about 1 - 2mm).
2. ** Pour into the gel solution **: When the agarose gel solution is cooled to about 50-60 ° C (temperature tolerable by hand), add an appropriate amount of nucleic acid dye (such as ethidium bromide, the final concentration is generally 0.5μg/ml), mix gently to avoid bubbles. Then slowly pour into the glue making mold to distribute the gel solution evenly.
3. ** Gel solidification **: Leave at room temperature for 30-60 minutes until the gel is completely solidified. At this time, the surface of the gel can be seen to be flat and elastic. Carefully pull out the comb to avoid damaging the loading hole.

Add sample
1. ** Prepare the sample **: Mix the sample to be tested with an appropriate amount of loading buffer. The loading buffer can provide a color indication to facilitate the observation of the migration of the sample in the gel, and at the same time increase the density of the sample so that it can sink into the loading well. Generally, mix the sample in a ratio of 4:1 or 5:1 to the loading buffer.
2. ** Sampling **: Put the solidified gel together with the mold into the electrophoresis tank, add 1 × TAE working buffer, so that the buffer does not pass the gel by about 1-2mm. Use a pipette to absorb an appropriate amount of the mixed sample and slowly add it to the loading well. Note that each loading well should not be overfilled to avoid sample overflow. At the same time, the nucleic acid molecular weight standard is added to the adjacent sample well as a reference for judging the molecular weight of the sample.

Electrophoresis
1. ** Connect the power supply **: Connect the positive and negative electrodes of the electrophoresis tank to the power supply. Note that the end of the sample hole is connected to the negative electrode, and the other end is connected to the positive electrode.
2. ** Setting parameters **: Set the appropriate voltage and electrophoresis time according to the gel concentration and sample characteristics. In general, 1% - 2% agarose gel is electrophoresis at 80 - 120V voltage for 30 - 60 minutes.
3. ** Observation of the electrophoresis process **: The migration of nucleic acid dyes can be observed during the electrophoresis process. When the indicator migrates to a suitable position (e.g. bromophenol blue migrates to about 1-2cm at the bottom of the gel), stop the electrophoresis.

RESULTS Observation and Analysis
1. ** Observation **: After the electrophoresis is completed, the gel is removed from the electrophoresis tank and observed under a gel imager or ultraviolet transmissimeter. Under ultraviolet light irradiation, the bands containing nucleic acids will fluoresce, and the sample bands and molecular weight standard bands can be clearly seen.
2. ** Analysis **: Estimate the molecular weight of the sample bands according to the position of the molecular weight standard bands and the known molecular weight size. At the same time, observe the clarity, brightness, etc. of the sample bands to judge the quality and concentration of the sample. If the band is blurred or there are multiple strains, it may indicate that the sample is degraded or impurities; if the band brightness is abnormal, it can be inferred that the sample concentration is too high or too low.