Casting a gel:
1) Measure out 0.4g of agarose into a weigh boat and add to flask
2) Add 50ml of TAE buffer
3) Melt in microwave for 1 minute, then cool under cold water or by adding cold TAE up to 50ml
4) Add 1.5 ul ethidium bromide (remember to throw tip in proper waste bucket)
5) Mix by swirling flask, and pour into casting frame
- To make casting frame, insert gel holder between bars on casting frame. Pull top bar down until level with holder. Turn screw to tighten. Add comb.
Running a DNA gel:
1) Add 1/10 volume of DNA loading buffer to sample
2) Remove gel from casting frame and place in gel tank
3) Add enough buffer to just cover the wells
4) Load sample in well
5) Load appropriate DNA ladder
6) Put cover on gel tank
7) Adjust voltage to 110V, then push “running man” to start the current
8) Stop gel when bottom loading dye band is 2/3 of the way down the gel
Extraction of DNA from gel fragment:
1) Look at gel briefly on high power UV box, take picture
2) Move gel to low power UV box (make sure gel box is clean and you wear the lovely UV shield)
3) Cut out gel fragment, dice it up, and move it to an eppendorf tube
4) Weigh the gel fragments in the eppe tube (make sure you zero the scale with an empty tube)- record how many milligrams the gel weighs
5) Add an equal volume of binding buffer to eppe tube with gel fragments (if gel weighs 100mg, add 100ul)
6) Melt at 55 degrees for about 5 minutes, or until you no longer see solid gel fragments
7) Add equal volume of isopropanol (100 ul) and mix well
8) Transfer contents of tube to purple column and spin at 14000 rpm for 30 seconds
9) Discard flow through
10) Add 500 ul of PB buffer, spin for 30 seconds at 14000 rpm, discard flow through
11) Add 750 ul of wash buffer, spin for 30 seconds at 14000 rpm, discard flow through
12) Centrifuge empty column for 1 minute at 14000 rpm
13) Remove dry column to new eppe tube without cap
14) Add 30 ul of EB buffer and spin for 1 minute at 14000 rpm
15) Remove eluted DNA to new eppe tube and test concentration on the nano drop