Pre-hybridization DNA preparation
A.) Reagents, Supplies, and Equipment
1.) Plant DNAeasy MaxiPrep kit Qiagen # 68163
2.) RNase with 1x RNase ONE Ribonuclease Promega Item # M4265
3.) Microcon YM-30 Concentrating Units, Millipore # 42409 OR #42410
4.) Nuclease-free Water, Ambion # 9934
5.) Sonicator capable of delivering 120 watts of ultra-sonic power directly to the
sample via a probe.
6.) Table-top centrifuge and rotor capable of 8000 x g.
B) Procedure
1.) Freeze one gram of plant fresh-weight tissue in liquid nitrogen and ground to a fine
powder. DNA was extracted using the Plant DNAeasy MaxiPrep kit (Qiagen),
substituting supplied RNase with 1x RNase ONE Ribonuclease (Promega) (50 Units
RNase/1 g fresh-weight tissue).
2.) Use a Microcon YM-30 concentrators to reduce the Qiagen prep’d DNA per sample
to approx. 200 µl.
3.) Add approx 300 µl nuclease-free H2O to bring total volume to approx. 500 µl.
Centrifuge @ 8000 x g for 10min. Ensure that the concentrators contain at least 500ul
before each 10min spin to avoid drying the sample against the membrane.
4.) Discard flow through and add approx. 300uL nuclease-free H2O. Centrifuge @ 8000 x
g for 10min (or a bit longer- you want approx 200uL DNA after this spin).
5.) Recover the concentrated sample by inverting the Microcon into a fresh collection
tube, and centrifuging @ 8000 x g for 2min.
6.) Make the volume up to 200 µl with Nuclease-free water. Lower volumes make
sonication difficult.
7.) Tune the sonicator as per the instruction booklet. Adjust the sonicator so that it
delivers 120 watts of power to the probe. Ensure everyone in the room has adequate ear
protection.
8.) Sonicate for 1:45 min.
9.) Check DNA purity (260/280=>1.8 and 260/230=>1.8) and concentration (at least 6ug
DNA in total). Dilute or concentrate sample till final DNA concentration =600ng/ul (+/20 ng/ µl).