The maximum volume of the sampled DNA is determined by the volume of the sample hole
The maximum amount of DNA that can be added to the sample hole depends on the size and number of DNA fragments in the sample. If a 5mm wide strip contains more than 500ng of DNA, it means that the loading hole is overloaded, which will cause tailing, "smile effect" and blurring on both sides of the strip. If the length of DNA increases, the above phenomenon becomes more serious.
When analyzing a single DNA sample (such as lambda phage or plasmid DNA), 100~500ng DNA can be added to each 5mm wide sample hole. If the sample is composed of many DNA fragments of different sizes (such as the digested sample of mammalian DNA), adding 20-30ug of DNA to each sample hole will not cause a significant decrease in resolution.
The maximum volume of DNA to be sampled is determined by the volume of the sample hole. The universal sample hole (0.5cmX0.5cmX0.15cm) can hold about 40μL of sample. Never fill the sample hole too full or even overflow. In order to avoid contamination of samples in adjacent wells caused by overflow, it is best to configure a slightly original gel to increase the volume of the sample hole or reduce the sample volume by ethanol precipitation and concentration of DNA.
Use a disposable pipette tip, automatic pipette, elongated Pasteur pipet (transfer pipet) or glass capillary to slowly add the sample mixture to the sample hole of the immersion gel. DNA molecular mass standards should be added to the two wells on the left and right sides of the sample well.
In many cases, it is not necessary to use a new pipette tip for each sample. It is only necessary to wash it with the buffer in the anode cell between two sample additions and then use it again. However, when the gel is to be used for Southern blot analysis or for experimental purposes to recover DNA bands, a new tip should be used for each sample.
Close the electrophoresis tank cover and connect the electrode plug. The DNA should swim towards the anode (red plug) side. A voltage of 1~5V/cm is applied, and the distance is measured from the anode to the cathode.
