DNA sequencing: Difference between revisions

==Gel Electrophoresis==
[https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis Agarose gel electrophoresis]
1.5% [https://en.wikipedia.org/wiki/Agarose agarose], 1.53 uL [https://en.wikipedia.org/wiki/Ethidium_bromide Ethidium Bromide] or 5 uL[https://en.wikipedia.org/wiki/GelRed GelRed] solution per gel, 75 mL 1x TAE[https://en.wikipedia.org/wiki/TAE_buffer buffer or LABTAE buffer].
To make a gel, add agarose to room temperature 1x TAE or LAB 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 Erlenmeyer flaskbeaker 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.
Optionally, you can use one of your lanes for a [https://en.wikipedia.org/wiki/Molecular-weight_size_marker DNA ladder] to verify that the gel is showing DNA correctly and see the size of your PCR product.
Run the gel at 100 volts DC for 20 minutes. If you leave it for 30 minutes or longer, you will lose your results. Higher voltages work more quickly but give bands that are less sharp.
Visualize the gel using a [http://www.instructables.com/id/UV-Transilluminator UV transilluminator]. Do not look at the UV light for more than a half second - you can use a sheet of glass to stop most of the UV and make it safer to view. Yellow safety goggles can be used to filter out the blue, making the results easier to see and providing additional UV eye protection. It is best to quickly photograph your gel and record your results from the photo to save your eyes and prevent sunburn.
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 - 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].