- DNA agarose gels Method: Work only on the EtBr bench, with nitrile (blue) gloves. 1. place the plastic tray in the electrophoresis tank and put the appropriate comb. 2. prepare a solution of agarose in TAE at a concetration appropriate for separating the DNA fragment size of your choise (see table) : Add powdered agarose to a measured TAE buffer in a glass tube, large gels 120 ml solution, small gels 50 ml solution. Close cap loosely and heat the slurry in a microwave until the agarose dissolves (gentle boiling, mix from time to time, be careful the bottle is hot!!!). Agarose concentration (%w/v) 0.3 0.6 0.7 0.9 1.2 1.5 2.0 Range of separation of linear DNA (kb) 5-60 1-20 0.8-10 0.5-7 0.4-6 (cut plasmid) 0.2-3 (shorter PCR products) 0.1-2 3. Cool the gel on the bench (until it is cool enough to hold the bottle in your hand for 5 seconds), and add EtBr to the bottle (0.5 µg/ml – for small gel one drop, large gel 2 drops). Close cap, mix well , and pour into the tray (insert comb!). 4. Allow the gel to set (30 minutes), fill the tank with TAE runnning buffer to cover the gel, and remove comb. 5. Mix sample with 6X loading dye, load marker (5 µl) and samples (small comb – 25-30 µl, large comb -60 µl, use about 100 ng DNA). 6. Run 20 minutes, 140 V: (The DNA will run towards the cathode (+) make sure you connect it to the bottom part of the gel). 7. Photograph your gel under UV. - DNA cont.Materials: 6 x Loading Dye (DNA) 25mg Bromophenol Blue (0.25%) 25mg Xylene cyanol FF (0.25%) 6ml Glycerol (60% v/v) Complete to 10ml with H2O (We use 6X DNA Loading Dye from Fermentas #R0611) EDTA 0.5M pH=8 186.1gr EDTA•H2O 800ml DDW add ~20gr NaOH (pellets), EDTA dissolves only close to pH 8.0 needs heating. Adjust to pH=8 with NaOH 10M and complete to 1L 50 x TAE (1L) 242gr Tris {1 x 40mM} 57.1ml Glacial acetic acid {1 x 40mM} 18.6gr EDTA {1x 1mM} (or 100 ml of 0.5M EDTA pH=8.0) Complete to 1L with H2O 10 x TBE (0.5L) 54gr Tris {1 x 89mM} 27.5gr Boric acid {1 x 89mM} 20ml EDTA 0.5M pH=8 {1 x 2mM) (optional: filter 0.45 µM to avoid precipitates) Complete to 500ml with H2O - DNA cont.DNA markers GeneRuler 1kb (Fermentas, SM0311) GeneRuler 100bp (Fermentas, SM0241) 6X DNA Loading Dye (Fermentas, #R0611) Add 6X Loading Dye and DDW according to table and store at 4 oC: DNA Ladder 6X Loading Dye Solution DDW 1 volume 1 volume 4 volumes