Running DNA Gels: Difference between revisions
Jump to navigation
Jump to search
PreDec2022>Thvu (→Goal) |
PreDec2022>Thvu |
||
Line 48: | Line 48: | ||
=Running the gel= | ==Running the gel== | ||
The higher the voltage, the faster the gel will run; but, the bands will be blurrier. We run gels between 40V (for publication images) to 80V-100V (for day-to-day gels). Be sure to watch the loading gel and not run your DNA off of the gel! | *The higher the voltage, the faster the gel will run; but, the bands will be blurrier. | ||
**We run gels between 40V (for publication images) to 80V-100V (for day-to-day gels). Be sure to watch the loading gel and not run your DNA off of the gel! |
Revision as of 20:54, 28 February 2021
Goal
- Goal: To successfully prepare agarose gel and run DNA gel electrophoresis.
Pre-Protocol Questions
- Do you have enough 1x TAE?
- If there is not enough 1x TAE, do you know how to make more?
- Do you know where the equipment in the lab is? (agarose, flasks, autoclave gloves, Ethidium bromide, etc...)
Special Reagents Needed
Creating 1X TAE
- If there is no 1X TAE, make it from the 50X TAE on the chemical shelf.
- Using a graduated cylinder to measure 1960mL MilliQ water, make a 40mL 50X TAE solution.
- Put this solution in a 2L bottle, screw the cap on, and mix well by shaking the bottle.
Agarose gels
Information
- We generally use 0.5% - 2.0% agarose gels; see the image below for more information on which percentage to chose.
- The agarose is found on the chemical shelf; it is different than the agar used to make plates. It is a more refined version of the same product.
- We use 150ml 1x TAE for the larger gels or 100ml 1x TAE for a smaller gel.
- Weigh out the correct amount of agarose and put in one of the smaller Erlenmeyer flasks.
- For a 1% gel, 1g in 100ml
- Add the correct amount to TAE and heat in the microwave.
- Our microwave is powerful — put it on power-level 6 for approximately 2 minutes, but watch it during this time. Do not allow it to boil for more than 3 seconds, as there is then a chance of it overflowing and creating a mess in the microwave.
- After it boils, use the orange autoclave gloves to take the flask out and gently swish it around.
- Is the agarose 100% clear and 100% melted? If there are bubbles, are they actively moving up? If not, it needs to be boiled for another 3-5 seconds until all of the agarose is melted.
Casting the Gel
- Prepare to cast the gel in the same boxes that we run the gel in: Put the tray perpendicular to how they are run, to create a seal on the open edges of the tray.
- Before agarose is added, add the combs to the gel.
- In most cases, it is perfectly OK to use two rows of combs in the gel, especially if you are expecting exactly one band of a predictable size.
- Wait for the agarose to cool down before casting the gel.
- Either leave the flask to cool, occasionally swishing it to mix the agarose, or carefully use an orange autoclave glove and stream tap water over the flask to cool it.
- Watch out — if the orange gloves become wet, they will conduct heat! Also, if the agarose cools down too much (if it becomes less than 100% clear, or has bubbles that are not actively rising), then the agarose needs to be re-melted in the microwave. Allow the flask to sit for 5-10 seconds, then test the temperature by touching it without the orange gloves. If it feels very warm, but not burning, then it is ready (~60 degrees).
- Add 1 uL Ethidium bromide (from the brown bottle in the refrigerator) per 100ml.
- Swirl the agarose to mix, but try to prevent bubbles from forming.
- Immediately carefully pour the agarose into the gel cast.
- Use a pipet tip to either pop any bubbles on the gel. You could use a pipet to vacuum up bubbles or move them to the side of the gel.
- Wait 10 minutes for the gel to cool
- To know if it cools completely, tap the gel tray. None of the agarose should shake.
Loading the gel
Running the gel
- The higher the voltage, the faster the gel will run; but, the bands will be blurrier.
- We run gels between 40V (for publication images) to 80V-100V (for day-to-day gels). Be sure to watch the loading gel and not run your DNA off of the gel!