Agarose Gels: Difference between revisions

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==== Protocol ====
==== Protocol<br> ====
'''For a 0.8% gel:'''


1.    Set up gel-casting dock.
===== For a 0.7% gel:<br> =====


2.    To a 250mL Erlenmeyer flask add 0.24 g of agarose (1 heaping green scoop) and 30 mL of 1X TAE
#Set up gel-casting dock.
 
#Add 0.14 g of agarose to a 250mL Erlenmeyer flask.  
3.    Microwave (30sec/15sec/15sec) – want the mixture to boil twice and become clear, swirl around.
#Add 400 µL of 50X TAE and 20 ml of ddH<sub>2</sub>O (from machine). Microwave (30sec/15sec/15sec) – want the mixture to boil twice and become clear, swirl around.  
 
#Pour hot mixture into gel-casting dock and place comb. Make sure no bubbles are formed. The gel will cast in 10-15 minutes in the fridge.  
4.    Allow the mixture to cool until you can place your hand on the bottom of the flask, add 3 uL of RedSafe DNA dye
#Remove the comb.<br>
 
#When taking out the gel, untighten the knob to release the top and bottom half of the casting dock, but hold them together with your hand. Carefully slide the gel out.<br>
5.    Pour hot mixture into gel-casting dock and place comb. Make sure no bubbles are formed. The gel will cast in about 45 mins at room temp or 15 minutes in the fridge.
#Place the gel into the gel-running dock and cover fully with running buffer. The running buffer for a gel: 0.5X TAE =&gt; (5 mL 50X TAE + 500 mL dH<sub>2</sub>O). Make sure the DNA is running towards the positive end!<br>
 
#Add ladder. For the small wells, add 2 μL of ladder; for the large wells, add 8 µL of ladder. The master-mix is located in the blue box in the -20°C freezer.<br>
6.    Remove the comb.
#Add the appropriate amount of 6x loading dye to the samples and then load into separate wells.  
 
#Run the gel at 100V for 45 minutes (35 minutes for smaller DNA &lt;1 kB).  
7.    When taking out the gel, untighten the knob to release the top and bottom half of the casting dock, but hold them together with your hand. Carefully slide the gel out.
#Stain the gel with ethidium bromide by adding the gel in the container labelled "Ethidium Bromide" and pour in all of the ethidium bromide from the 5x container. Put the container on the shaker for 5-10 minutes. Pour the ethidium bromide back into the container after use.<br>
 
#Rinse the gel twice with tap water and let the gel soak in water for 5-10 minutes.
8.    Place the gel into the gel-running dock and cover fully with running buffer. The running buffer for a gel: 0.5X TAE => (10 mL 50X TAE + 990 mL dH<sub>2</sub>O). Make sure the DNA is running towards the positive end!
#Pour the water out into the sink.<br>
 
#Place the gel on the UV imaging plate and place the plate into the gel imaging doc.  
9.    Add ladder. For the small wells, add 2 μL of ladder; for the large wells, add 6 µL of ladder. The ladder can be found in the ladders box in the -20°C freezer.
#Turn the computer on and sign in.  
 
#Open the program "Image Lab" and press select, then "nucleic acid", then "ethidium bromide".<br>
10. Add the appropriate amount of 6x loading dye to the samples and then load into separate wells.
#Press "run protocol".  
 
11. Run the gel at 100V for 45 minutes (35 minutes for smaller DNA <1 kB).
 
12. Place the gel on the UV imaging plate and place the plate into the gel imaging doc.
 
13. Turn the computer on and sign in.
 
14. Open the program "Image Lab" and press select, then "nucleic acid", then "ethidium bromide".
 
15. Press "run protocol", alternatively press the green button on the GelDoc after opening Image Lab.


<br>
<br>

Revision as of 15:07, 25 February 2022

Protocol

For a 0.7% gel:
  1. Set up gel-casting dock.
  2. Add 0.14 g of agarose to a 250mL Erlenmeyer flask.
  3. Add 400 µL of 50X TAE and 20 ml of ddH2O (from machine). Microwave (30sec/15sec/15sec) – want the mixture to boil twice and become clear, swirl around.
  4. Pour hot mixture into gel-casting dock and place comb. Make sure no bubbles are formed. The gel will cast in 10-15 minutes in the fridge.
  5. Remove the comb.
  6. When taking out the gel, untighten the knob to release the top and bottom half of the casting dock, but hold them together with your hand. Carefully slide the gel out.
  7. Place the gel into the gel-running dock and cover fully with running buffer. The running buffer for a gel: 0.5X TAE => (5 mL 50X TAE + 500 mL dH2O). Make sure the DNA is running towards the positive end!
  8. Add ladder. For the small wells, add 2 μL of ladder; for the large wells, add 8 µL of ladder. The master-mix is located in the blue box in the -20°C freezer.
  9. Add the appropriate amount of 6x loading dye to the samples and then load into separate wells.
  10. Run the gel at 100V for 45 minutes (35 minutes for smaller DNA <1 kB).
  11. Stain the gel with ethidium bromide by adding the gel in the container labelled "Ethidium Bromide" and pour in all of the ethidium bromide from the 5x container. Put the container on the shaker for 5-10 minutes. Pour the ethidium bromide back into the container after use.
  12. Rinse the gel twice with tap water and let the gel soak in water for 5-10 minutes.
  13. Pour the water out into the sink.
  14. Place the gel on the UV imaging plate and place the plate into the gel imaging doc.
  15. Turn the computer on and sign in.
  16. Open the program "Image Lab" and press select, then "nucleic acid", then "ethidium bromide".
  17. Press "run protocol".


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