Agarose gel electrophoresis

Materials

TAE buffer (1x) 4.84 g Tris-Base, 1 mM EDTA, 1.14 mL acetic acid, fill 1 L with A.bidest. Agarose

Procedure

• Weigh in 2.1 g agarose for a 0.7% gel or 4.2 g for a 1.4% gel and transfer to a 500 mL bottle
• Add 300 mL 1xTAE
• Heat up in microwave (Put the cap loosely on the bottle! Beware of boiling delay, which can lead to explosive discharge of hot liquid, which will easily burn your hands). Therefore, swirl repeatedly in order to solve agarose completely and avoid boiling delay
• Let dissolved agarose solution cool down to 50°C
• Prepare agarose gel casting stand by inserting the gel tray with 90º in the gel chamber, insert comb(s), and adjust with bubble level
• Pour lukewarm agarose into gel tray and let the gel solidify
• work out pipetting scheme and take out the comb(s)
• Mix samples with 6x sample buffer (GEBS buffer) and pipette according to the scheme
• Run the gel at 90V (analytical) or 50V (preparative) or 20V (preparative, overnight)
• Cut used part of the gel with a scalpel and stain in staining bath
• Destain in distilled water
• Take picture with GelDoku and save the image file on your folder on the file server
• For preparative gels: cut out the desired band with the scalpel (on the small UV-table, right next to the Geldoku) and place gel slice in 2 mL Eppendorf tubes.