Team:Queens Canada/Notebook/Protocols/GelElectrophoresis
From 2011.igem.org
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<regulartext> <b> Electrophoresis </b> </regulartext> <br> | <regulartext> <b> Electrophoresis </b> </regulartext> <br> | ||
- | <regulartext>1. Add | + | <regulartext>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. <br> |
- | + | 2. Add 2μL of loading dye into each well. <br> | |
+ | 3. Fill the gel box with TBE until the entire gel is immersed in solution. <br> | ||
+ | 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. <br> | ||
+ | 5. Close the lid of the gel box. Run the gel at 100V constant voltage for 1 hour. <p> | ||
</regulartext> | </regulartext> | ||
+ | <regulartext> <b> Imaging </b> </regulartext> <br> | ||
+ | <regulartext>1. Turn off the gel box power supply. <br> | ||
+ | 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. <br> | ||
+ | 3. Log in to the computer. <br> | ||
+ | 4. Open Genesnap. Click the big green button on the left to take a picture, and manipulate it with the sliders on the right. <br> | ||
+ | 5. Print out the gel picture, label each lane, and paste it into your lab book. <br> | ||
+ | 6. Save the picture (in .tif format) in the QGEM folder on the USB stick. <br> | ||
+ | 7. Upload the picture to Google Docs.<br> | ||
+ | 8. Remove the gel and dispose of it in the proper waste bin. Wipe down the imaging machine and lock the dark room door. <p> | ||
+ | |||
+ | </regulartext> | ||
Latest revision as of 20:33, 28 September 2011
![Queen's](https://static.igem.org/mediawiki/2011/c/c6/Queens_Canada_Test2.png)
![Queen's](https://static.igem.org/mediawiki/2011/2/21/Queens_CanadaTitleProtocols.png)
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.
2. Use appropriate volume of ladder (depends on the ladder used).
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.
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.