Virginie Orgogozo
Preparation of genomic DNA
Using DNeasy Tissue Kit QIAGEN #69506
(p29-23)
very good to amplify fragments that are 2-10kb long, good for samples
stored in ethanol
if you start from living
material:
1. add 180uL PBS into a 1.5mL microcentrifuge tube.
2. place up to 50mg of male+female flies (20 flies = 40mg) in the tube.
if you start from 3-5 flies
stored in ethanol:
1. add 180uL PBS into a 1.5mL microcentrifuge tube.
2. dry the flies on tissue for a few seconds and then place them into
the tube+PBS.
3. homogenize the sample using an electric homogenizer or a disposable
microtube pestle.
4. add 10uL of RNAse (20mg/mL, stored at-20∞C) and incubate for 2min at
RT.
5. add 20 uL proteinase K and 200uL buffer AL to the sample, mix
thoroughly by vortexing, and incubate at 70∞C for 10 min.
6. add 200uL ethanol (96-100%) to the sample, and mix thoroughly by
vortexing. A white precipitate may form.
7. pipet all the mixture from step 3 (including any precipitate) into
the DNeasy spin column placed in a 2mL collection tube (provided).
Centrifuge at 8000rpm for 1min. Discard flow-through and collection
tube.
8. place the DNeasy spin column in a new 2mL collection tube
(provided), add 500uL buffer AW1 and centriguge 1min at 8000rpm.
Discard flow-through and collection tube.
9. place the DNeasy spin column in a new 2mL collection tube
(provided), add 500uL buffer AW2, and centrifuge for 3min at full speed
to dry the DNeasy membrane. Discard flow-through and collection tube.
10. place the DNeasy spin column in a clean 1.5mL or 2mL
microcentrifuge tube and pipet 100uL (or 50uL if you start from 3-5
flies) buffer AE directly onto the Dneasy
membrane. Incubate at RT for 1min, then centrifugate for 1min at
8000rpm to elute.
11. pipet 100uL (or 30uL if you start from 3-5 flies) buffer AE again
onto the Dneasy membrane, incubate at
RT for 1min, and centrifugate for 1min at 8000rpm to elute.
12. check 10uL on a 0.8% agarose gel