TITLE: Honeybee DNA extractions in 96-well plates
AUTHORS: Tom Harrop, Reuben McKay Vercoe

[STEPS]
?. [Prepare lysate for extraction]
Prepare lysate for extraction
?. [Prepare lysate for extraction]
Homogenise the tissue for 90 seconds at 20 Hz using a Mixer Mill or Tissue Lyser.
?. [Prepare lysate for extraction]
Prepare 300 µL lysis buffer per well.  AB1Lysis solution287 µL2RNase solution (10 mg/mL)3 µL3Proteinase K solution (20 mg/mL)10 µL4Total300 µLAdd the 300 µL lysis buffer to each well of tissue plate.
AB1Lysis solution287 µL2RNase solution (10 mg/mL)3 µL3Proteinase K solution (20 mg/mL)10 µL4Total300 µL
?. [Prepare lysate for extraction]
Spin the plate for 10 minutes at 1,000 RPM.
Centrifuge: 1000 33, 10 min, 15 10
?. [Prepare lysate for extraction]
Seal the plate and mix on the plate shaker. Make sure the paste is resuspended.Incubate at 55°C for 150 minutes, shaking at 1000 rpm (or shake the plate for one minute every 30 minutes).
55 °C
?. [Prepare lysate for extraction]
Transfer 200 µL of lysate to a new extraction plate.
?. [Prepare lysate for extraction]
Dissect e.g. 8 drone heads for a single sample.Pool the dissected material into a 2 mL safe-lock tube.Add 4 3.2 mm stainless steel ball bearings and 500 µL PBS.
?. [Prepare lysate for extraction]
Centrifuge the homogenate for 10 minutes at 16,000 g and remove and discard the top layer (i.e. PBS).
Centrifuge: 16000 34, 10 min, 21 10
?. [Prepare lysate for extraction]
Transfer about 150 mg of the tissue paste to a 96-well plate for lysis.The remaining paste can be stored at -80°C and used for subsequent extractions.
?. [DNA extraction]
Set up the reagents from the ZYMO Quick-DNA Magbead Plus Kit
?. [DNA extraction]
Add the following reagents to the 7-position ReservoirRack  ABCDEFGHIJKLM1ReagentReservoir
volume (mL)81624324048566472808821: (490 µL MBB : 10 µl beads)1004657865712657166572065724657286573265736657406574465732: empty











43: DNA pre-wash buffer10078571505722257294573665743857510575825765457726577985754: g-DNA wash buffer10078571505722257294573665743857510575825765457726577985765: g-DNA wash buffer10078571505722257294573665743857510575825765457726577985776: Water (for rinse)10070571345719857262573265739057454575185758257646577105787: Tris-HCl1011831583198323832783318335833983438347835183
ABCDEFGHIJKLM1ReagentReservoir
volume (mL)81624324048566472808821: (490 µL MBB : 10 µl beads)1004657865712657166572065724657286573265736657406574465732: empty











43: DNA pre-wash buffer10078571505722257294573665743857510575825765457726577985754: g-DNA wash buffer10078571505722257294573665743857510575825765457726577985765: g-DNA wash buffer10078571505722257294573665743857510575825765457726577985776: Water (for rinse)10070571345719857262573265739057454575185758257646577105787: Tris-HCl1011831583198323832783318335833983438347835183
?. [DNA extraction]
Run the epMotion protocol 88x Quick-DNA Magbead Plus premixed beads DNA rinse.Because the rinse step is programmed once for each plate column, the protocol has to be copied and modified for each run.
?. [DNA extraction]
The master program is at Dearden/Tom/Quick-DNA Magbead Plus/88x Quick-DNA Magbead Plus premixed beads DNA rinse.Copy that file to your Runs directory and change the name. Use a name that includes today's date.
?. [DNA extraction]
Change the sample number at steps 1 and 55.Delete rinse steps between 42 and 52, leaving only the steps for columns that have samples in them.
?. [DNA extraction]
Run the protocol with level sensing enabled.