Brandon:Protocols/agarose gel protocol

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agarose gel protocol[edit]

1. If needed sterilize in UV box the clear plastic gel holding tray, opaque plastic tray that holds the clear plastic holding tray, and the gel comb for 15 minutes.


2. determine the percentage of agarose gel to add and add the specified amount of agarose to the gel.

  • for 85 bp fragment 4.5% agarose was used.


3. pour agarose into flask with 0.5X TBE and swirl until all agarose is unclumped. Set hotplate to ~220C and stir with a stirbar in the flask. Cover the top of the flask with tinfoil so evaporation does not occur. Can also use microwave (no tinfoil)

0.5X TBE (mL)  gel size
60             full or compartmentalized full
40             half


4. Once agarose has dissolved into solution (for 4.5% usually when solution is boiling, ~10 minutes) then turn off the heating plate and let cool some, until some of the bubbles have popped.


5. Once cooled some (but still hot), add SYBR-safe to liquid agarose and stir quickly until ubiquitously in the solution. 10,000X so .1 uL per mL of liquid agarose. (60 mL then 6 uL SYBR-safe)


6. Can let cool more if needed, then pour agarose into assembled gel holder (clear plastic gel holding tray, opaque plastic tray that holds the clear plastic holding tray, and the gel comb)


7. Ensure solution is evenly distributed around in the gel holder, and let sit until hard and then remove gel comb. Can put in 4C to cool faster.


8. Fill/(wash if needed) gel box with 0.5X TBE, and put in the clear plastic gel holder holding the solidified gel in, and add 0.5X TBE if needed so the gel is lightly covered by 0.5X TBE. Then proceed to add ladder and samples, and run gel until bands migrate at least half way down. ran at 135V for 60 mins with 4.5% agarose.(always run to RED)


9. After run is finished, gel is now readying for gel size selection or imaging on gel dock. (NOTE, UV from gel doc damages DNA)