Protocol for Extractions using Qiagen Kit
Butterfly Leg Extractions
Materials:
Glass bottle with wick in ethanol
Forceps
Blue tubes (specially made for extractions)
Blue pestles (found with blue tubes)
Qiagen Kit for Butterflies (contains all buffers needed)
Qiagen DNEasy Blood and Tissue Handbook
Proteinase K and RNAse (on shelf)
Water bath at 56°C
Centrifuge
1) Label appropriate number of 1.5mL tubes with unique DNA numbers.
2) Get out glass bottle with ethanol in it (sitting on bench – has wick with a glass
cover on top). Light with lighter.
3) Run forcep tips through flame to sterilize.
4) In blue binder, record unique DNA number, species, sex, place of collection, date
collected (if known), male/female (if known)
5) Take one leg from each butterfly with forceps
a. Grab from base of body – each leg will have 2 sections
b. Place leg in separate 1.5mL tube
c. Close tube to prevent any contamination
d. Sterilize forceps with flame
e. Repeat
6) Using one pestle for each tube, crush leg by pushing pestle all the way down into
tube, and spinning while applying pressure. You should see the leg splitting,
revealing a white substance (the muscle of the leg). Leave pestle in tube.
7) Following July 06 Qiagen spin column protocol (p. 28), place 180uL ATL buffer
into each tube.
8) Using pestle, crush legs a bit more in buffer.
9) Take out pestle, making sure no large amounts of tissue are on it.
10) Add 20uL Pro K to each tube – vortex to mix. Place all tubes in 56°C bath for at
least 3 hours.
a. Can go overnight if need be.
b. Usually, the mixture is a brown color when done – you should see no
white residue, just the black exoskeleton of the leg.
11) Add 4uL RNAse to each, mix by vortexing and let sit for 2 minutes at room temp.
12) While incubating, mix 200uL Buffer AL with 200uL 100% ethanol (scale up
appropriately depending on how many samples you have i.e. 2 samples would be
400 of each, etc.). Mix by vortexing.
13) While incubating, get out appropriate amount of spin columns, one each for
sample. Label tops with same labels as above. Also get out 2 collecting tubes for
each spin column (in bags in the kit itself).
14) Vortex each sample for 15 seconds, then add 400uL of the AL/Ethanol mixture to
each. Vortex to mix.
15) Transfer mixture to spin columns (usually set the 1000uL pipet to ~650uL –
that’ll get most of it.)
16) Centrifuge at 8000rpm for 1 min. DNA will stick in the silica of the spin column.
17) Take spin column out of collection tube – discard the flow-through in TAE waste
bucket in hood. Place spin column in new collection tube.
18) Add 500uL Buffer AW1 to each. Spin at 8000rpm for 1 min.
19) Place spin column in new collection tube. Discard flow-through in TAE bucket in
hood.
20) Add 500uL Buffer AW2 to each. Spin at 14,000rpm for 3 min.
21) While spinning, label regular microcentrifuge tubes with unique labels (same as
above).
22) Once done spinning, carefully remove spin column from collection tube.
a. You want to avoid touching the bottom of the spin column to the ethanol
flow through in the collection tube.
b. If it does touch, empty the collection tube, place spin column back in,
centrifuge at 14,000rpm for 1min.
23) Place the spin columns in their labeled microcentrifuge tubes.
24) Pipet 50uL AE buffer into each – try to get the drop in the center of the column.
Incubate for 1 minute at room temp.
a. If two legs used, double elution volume to 100uL.
25) Centrifuge at 8000rpm for 1 min. to elute DNA.
26) Place DNA in 4°C refrigerator for use.
Gel Check
1) Make a 1% DNA gel
a. Small: 0.4g agarose in 45mL 1X TAE + 1uL GelRed
b. Medium: 0.8g agarose in 80mL 1X TAE + 1.5uL Gel Red
c. Large: 1.5g agarose in 150mL 1X TAE + 2.5uL GelRed
2) Load 4uL DNA with 1.2uL of 6X Orange Loading Dye. (~5uL load)
3) Load 1.2uL Invitrogen TrackIt Ladder per lane needed.
4) Genomic DNA should appear very high up on the gel, just one band.
5) Label picture and place in blue binder alongside descriptions.