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