Sheffield Molecular Genetics Facility Assessing the quality and quantity of DNA (using a 0.8% agarose gel and fluorometer) Making an agarose gel 1) Place masking tape at the ends of a 20cmx20cm plastic gel tray. Place four 27well combs in position. Place on a flat surface 2) Make up a 0.8% agarose gel (250ml 1xTBE, 1.75 g agarose) in a 500ml flask. Heat in microwave (NB. take care as it becomes superheated. Stir every minute to release the bubbles). Add 9ul ethidium bromide (1mg/ml) and cool under a tap whilst rotating the flask 3) Pour into the prepared plastic gel tray 4) Leave to set for approx. 30 minutes 5) When set remove combs and place in electrophoresis tank containing 1XTBE buffer Preparing samples to load on an agarose gel 1) Pipette 14ul 1x Orange G loading buffer into wells into a 72-well Terrasaki plate 2) Add 1ul of the sample 3) To the last well add 0.5ug of Lambda DNA standard 4) Load all the sample on the gel 5) Electrophorese the gel (100Volts, DNA is –ve so goes toward the +ve electrode). Sheffield Molecular Genetics Facility Quantification of DNA using a DNA fluorometer (Hoefer DynaQuant200) 1) Switch on the fluorometer and allow it to warm up for 20 minutes 2) Add 2ml of fluorometer dye solution to a clean cuvette. Place in the fluorometer 3) Press “Zero” 4) Remove the cuvette and add 2ul of 100ng/ul calf thymus DNA 5) Place in the fluorometer, press CALIB, type in 100 then press ENTER. The value should read “100” 6) Remove the cuvette, rinse with 2ml of fluorometer dye solution 7) Add 2ml of fluorometer dye solution to the clean cuvette. Place in the fluorometer 8) Press “zero” if necessary. Add 2ul of your sample. 9) Read off the value given as ng/ul. This is the DNA concentration of your sample 10) Repeat from step 5 with all your samples 11) You can now dilute all your samples to “PCR concentration” i.e. 10ng/ul by removing 10ul of sample to a new tube and adding as water the concentration in ng/ul –10. This will make a 10ng/ul stock i.e Fluorometer value of 210. Remove 10ul and place in a tube, add 200ul of water. Mix. This is makes a 10ng/ul working solution.