Team:Queens Canada/Notebook/Protocols/GelElectrophoresis

Storage and Labelling
- Label the file of picture of gel according to this standard:
- [#] P-dd_mm_yy
- # is the nth gel run that day in the lab
- P is the initial of the last name of the professor whose lab the gel was run in

Materials
- Loading Dye
- 100kb+ DNA Ladder
- 5X T4 DNA Ligase (4µL)
- 1% Agarose Gel (50mL 1X TBE and 0.5g Biotech Grade Agaraose)
- Gel box and power supply
- Ethidium bromide stain
- UV box/gel imager

Making Agarose Gel
1. Weigh out 0.5g of agarose and add to a 250ml Erlenmeyer flask.
2. Add 50ml 1X TBE to the flask and mix by swirling.
3. Microwave TBE agarose until the solution becomes clear and obtains a uniform consistency. (First microwave for 1min and then for 30sec intervals. DO NOT allow the solution to boil over in the microwave).
4. Use glove to remove flask from the microwave. Allow flask to cool on the lab bench for 5 min (But do not wait until the gel starts to polymerize)
5. Take out EtBr from -20⁰C freezer. Add 3μL to 50mL TBE agarose and swirl the flask to mix. Return EtBr to the freezer immediately after use. (Note: EtBr is a carcinogen and a mutagen. Always use glove and lab coat, if available, to handle things contaminated with EtBr.)
6. Carefully pour TBE agarose into the casting tray to avoid bubbles. Make sure the tray is placed on a flat surface. Insert comb into the TAE agarose gel.
7. Let the gel polymerize for 20min.

Sample Preparation
1. Add appropriate amount of loading dye such that the mixture of loading dye and sample contains 1X concentration of dye. If using 5X dye, use 4 parts sample and 1 part dye.
2. Use appropriate volume of ladder (depends on the ladder used).

Electrophoresis
1. Once the gel is solidified, remove the comb carefully and place the casting tray in the gel box. Make sure the wells point towards the black (negative) electrode.
2. Add 2μL of loading dye into each well.
3. Fill the gel box with TBE until the entire gel is immersed in solution.
4. Load prepared samples into the wells. Slowly pull out the pipette tip from the well before releasing the piston of the pipette. This avoids inserting bubbles into the wells, which will disturb the sample.
5. Close the lid of the gel box. Run the gel at 100V constant voltage for 1 hour.

Imaging
1. Turn off the gel box power supply.
2. Transport the gel in a plastic box to the dark room. Bring two sets of gloves if you are doing this by yourself, because you cannot touch the computer mouse with EtBr contaminated gloves. The dark room key (with attached USB key) is in the top drawer of the gel box bench.
3. Log in to the computer.
4. Open Genesnap. Click the big green button on the left to take a picture, and manipulate it with the sliders on the right.
5. Print out the gel picture, label each lane, and paste it into your lab book.
6. Save the picture (in .tif format) in the QGEM folder on the USB stick.
7. Upload the picture to Google Docs.
8. Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door.