====== Gel Electrophoresis ======

 --- //[[grabbermop@gmail.com|Olivia Heath]] 2018/05/01 16:28//

==== Protocol ====
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**Setting up a Gel**
Decide how many wells you are going to need. We have 12, 20, and 25 well combs. Keep in mind that you will need to put HL on either side of your samples. You will also need to make your asymmetrical so as to avoid confusion. If your gel is symmetrical, you will need to grab the XACTO knife and cut off the top right corner.


**You Will Need**
  * 2% Agarose - 2.5g for one gel.
  * 125mL TAE for 2% Agarose

**Procedure**
**DO NOT USE A STIR BAR, WEAR GLOVES**

STAGE 1
  - Mix 2.5 g agarose with 125mL TAE in a 250mL flask. Microwave for 30 sec, swirl, repeat. Do this until the mixture is completely clear. 
  - Get large gel box and take off lid. Set orientation of plate so orange strip is on the top and bottom. Push down. Wait for the flask to cool. Put the comb in. For one well, put it on the top. For two, put it on 1 and 3. Never put the comb in position two or four. 
  - Wait for your gel to completely solidify- becoming opaque in all areas. Gently take the comb out. Flip the gel so the orange strip is on the left and right, making sure your comb is on the left (gels run from black (anode) to red(cathode)). Fill with TAE to fill line.

STAGE 2
  - Add 6x loading dye (Drawer 36) to your samples. For 5ul of sample, add 1ul loading dye.
  - Use 5ul of Hyper Ladder (23kb or 6 kb), in DNA ladder box in the -20. 
  - Gently pipette 5ul of your HL and samples with loading dye into the wells.
  - Plug the red plug into the red jack and the black plug into the black jack.
  - Turn the machine on. You should see the machine reach 160 volts (+/- 10 volts).
  - Wait until the electrophoresis line runs 3/4 the way down your gel.

STAGE 3
  - Put on gloves. EtBr causes cancer.
  - Turn off machine and take off the gel box cover.
  - Place the gel in a glass tupperware container. 
  - Cover the gel with the TAE from the gel box.
  - Pipette 5ul Ethidium Bromide to both sides of the gel.
  - Rock the tupperware gently.  
  - Wait 45 minutes to complete the staining of the DNA on the gel.
  - Remove liquid from tupperware, diposing of it in the plastic containers provided. Do not put it down the sink.
  - Cover the gel with dH2O
  - Wait 15 min.
  - Visualize with the Chemidock in the AIM core or in the Liu lab.