DNA sequencing: Difference between revisions

[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis]
1.5% [https://en.wikipedia.org/wiki/Agarose agarose], 31.5 uL [https://en.wikipedia.org/wiki/Ethidium_bromide Ethidium Bromide] or [https://en.wikipedia.org/wiki/GelRed GelRed] solution per gel, 75 mL 1x [https://en.wikipedia.org/wiki/TAE_buffer TAE buffer].
To make a gel, add agarose to room temperature 1x LAB or TAE buffer, then heat in the microwave to dissolve it into solution. It is important to make it completely clear - no grains of agarose should be visible when you swirl the beaker in front of a light. This often involves a couple minutes of boiling with the microwave - run the microwave until it boils, stop it before it boils over, swirl and run it again every few seconds for a couple minutes. Be careful not to fill the beaker too full or it can get superheated and boil over, possibly burning your hand. Once the gel is completely dissolved, add 3 uL Ethidium Bromide solution per 75 mL gel.
To use the gel, put it into a [http://citizensciencequarterly.com/2011/10/cheapass-science-gel-box/ gel box] and pour 1x TAE buffer over it until the buffer barely overflows to cover the gel, making sure to fill all the wells as you pour the buffer. Keep in mind that the DNA will move towards the positive electrode, so make sure you orient the gel correctly. Set a small sheet of parafilm in front of the gel box and pipette 3 uL drops of 10x loading buffer for as many wells as you have in your gel. Do this in rows of 8 so it is easy to keep track of which sample is going into each lane. Add 8 uL of your PCR products to the drops on the parafilm and pipette them into the wells on the gel. This process should be done quickly so the drops don't evaporate too much and the DNA does not disperse throughout the gel before you turn on the electricity. You don't need to hurry, but you also shouldn't stop in the middle and go do something else for awhile.
If you get a strong or medium-strong clear band, it is very likely that you will get a clean sequence from the sample. If you get a weak band, smear or no band at all, [https://en.wikipedia.org/wiki/Nested_polymerase_chain_reaction nested PCR] can be used to further amplify the DNA.
TAE buffer is the standard buffer used, but it may not be the best choice - sodiumBorax (Sodium borate) can be used instead, allowing higher voltage and therefore faster gels. See [http://bitesizebio.com/25078/faster-even-cooler-dna-gels/ Faster even cool DNA gels].
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