Magni:Protocols/Electrophoresis

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(TBE 5X)
Current revision (04:26, 3 October 2012) (view source)
 
Line 37: Line 37:
** 54 g of Tris;
** 54 g of Tris;
** 27.5 g of Boric acid;
** 27.5 g of Boric acid;
-
** 20 ml of EDTA 0.5 M.
+
** 20 ml of EDTA 0.5 M, pH 8.0.
* Mix with a magnetic stirrer until dissolved.
* Mix with a magnetic stirrer until dissolved.
* Bring volume to 1 L with deionized water (blue tap from the Millipore machine).
* Bring volume to 1 L with deionized water (blue tap from the Millipore machine).

Current revision

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Electrophoresis

This is the procedure to prepare and run a 1% agarose gel. According to the number of samples to run, different gel sizes can be prepared: small gel (8 wells), medium gel (15 wells), large gel (30 wells).

GEL PREPARATION (ALWAYS WITH GLOVES!)

  • Prepare a clean gel tray (take it from the 3rd drawer under the pH-meter or near the sink) by tightly closing the extremities with tape.
  • Put a proper comb on the tray.
  • Weight agarose powder in a clean flask (0.5/1.5/2.5 g for small/medium/large gel).
  • Measure 50/150/250 ml (for small/medium/large gel) of TBE 1X with a cylinder and pour it into the flask.
  • Close the flask with tissue and boil in microwave oven (maximum power) until agarose is melted.
  • Wait until the flask cools down (one should be able to handle it without oven gloves).
  • Add 1/2/3 ul (for small/medium/large gel) of ethidium bromide (stored protected from light), mix well and pour into the tray.
  • Let the gel polymerize at room temperature.

GEL RUN: (ALWAYS WITH GLOVES!)

  • Add Blue Juice 6X to the DNA sample (e.g. 5 ul of Blue Juice and 25 ul of DNA mix).
  • Remove tape, put the tray with the gel in an electrophoresis chamber of proper size and remove the comb to form the wells (gel must be oriented with wells near the black wire connection).
  • Add TBE 1X to the chamber until the gel is completely submerged (if TBE 1X is clear, it can be re-used up to 5 times).
  • Pipette 6 ul of Marker (Fermentas GeneRuler 1 kb Plus DNA ladder) in a well.
  • Pipette up to 35 ul of DNA with Blue Juice into the other wells.
  • Close the chamber, turn the power supply on, set it to 90 V and press "run" (check if the blue dye runs in the right direction!). Voltage can be increased up to 130 V when in a hurry.
  • When the blue dye reaches half of the gel, the run could be stopped. Transfer the gel on the transilluminator and turn it on to check the DNA bands. Set the UV power to 70% if the DNA has to be cut and gel-extracted. Set it to 100% otherwise.

SAFETY NOTES:

  • ethidium bromide is toxic and mutagenic and is a suspected carcinogen. Be careful when handling ethidium bromide solutions (always with gloves, as described above) and when fumes come out from the flask during gel preparation.
  • ultraviolet radiation is harmful to the skin and eyes. Wear gloves and a face shield when using an exposed ultraviolet source. Always use the transilluminator protection.

TBE 1X

  • Mix 200 ml of TBE 5X and 800 ml of deionized water (blue tap from the Millipore machine).
  • Store at room temperature.

TBE 5X

  • For 1 L of TBE 5X, add to a clean becker:
    • 800 ml of deionized water (blue tap from the Millipore machine);
    • 54 g of Tris;
    • 27.5 g of Boric acid;
    • 20 ml of EDTA 0.5 M, pH 8.0.
  • Mix with a magnetic stirrer until dissolved.
  • Bring volume to 1 L with deionized water (blue tap from the Millipore machine).
  • Transfer into a plastic bottle and store at room temperature.
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