Difference between revisions of "DeRosa:Protocols/Agarose gel"
(New page: Supplies Agarose Low Melting [Fisher] TBE Deionized water Ethidium Bromide solution (Made from Fisher Biotech Ethidium Bromide) [Promega] 25 base pair ladder [Promega] Blue/Orange 6X Gel l...)
Latest revision as of 22:49, 1 October 2012
Supplies Agarose Low Melting [Fisher] TBE Deionized water Ethidium Bromide solution (Made from Fisher Biotech Ethidium Bromide) [Promega] 25 base pair ladder [Promega] Blue/Orange 6X Gel loading dye
Thermometer Erlenmeyer flask Microwave Agarose gel set tray & loading system: Owl Model A1 Gator Gel System (Large) OR i-Mupid mini gel 10uL pipetteman (Fisher or Gilson) & gel loading tips
AVOID The following -Taping the combs to be used as a wall of some sort, the sticky stuff stays stuck and ruins the comb -Allowing to gel to sit outside for long in warm weather -Do not prepare gels more than a few hours ahead of time (storing in fridge seems to produce poor results)
General Instructions: -For the mini gels use a 2.5% for the large gels look up in manual which percent to use for base pair size (generally used 2%) -Wear nitrile gloves as EtBr is toxic -Prepare a general 10mg/ml solution of Ethidium Bromide -Large Gel system instructions saved on V:\shared\DeRosaGroup\Manuals\Horiz Electro gel Owl.pdf -Small -iMupid gel system uses 50V, large Owl gel system uses 160V
- Weigh out desired amount of Agarose Low Melting (% of total gel volume)
- Add 1X TBE (Diluted accordingly with deionized water) and agarose to a large Erlenmeyer flask (at least 3 times the size of the volume of your solution). Plug the opening with a paper towel.
- Microwave for 1-3 minutes (depends on volume) until all agarose has dissolved without it boiling over
- Allow agarose solution to cool at room temperature until 60˚C (measure with a thermometer. Do not allow to cool much below 60˚C.)
- Add EtBr to a concentration of 0.5 ug/mL
- Slowly pour into gel tray (with the appropriate comb already inserted)
- Allow gel to solidify (Approximately 1 hour for a large gel)
- In the meanwhile, prepare loading samples in a small centrifuge or PCR tube and label
a. 1uL loading dye : 5uL DNA sample (or appropriate ladder)
- Once solidified, place gel with tray into system, remove combs and end guards and fill the system with TAE buffer to fill line. TBE buffer is preferred for large gels.
- Load 5uL of sample into the wells with gel loading barrier tips and a 10uL pipetteman
- Plug in appropriate power supply
- Run gel run
- Turn on Computer connected to Alpha Innotech gel visualizer
- Visualize on Alpha Nanotech UV gel visualizer, making sure the filter was on EtBr