Arabidopsis DNA extractions: 96-well format:
(Alonso-Stepanova Lab protocol)
1. Place metal beads into the wells of a 96-well plate (one bead per well). Mark one side of the plate with a Sharpie to keep track of the orientation and order of the strips in the rack (draw a diagonal line across the sides of 12 tubes in position A).
2. Collect tissues (one leaf or 25-50 3-day-old seedlings per well), cap the strips, cover the plate with a lid, and store at -80C until ready to grind.
3. Grind frozen (right out of -80ºC) samples in a Qiagene shaker (remove the lid, shake at setting 20 for 1min, rotate plate 180 degrees, and repeat). Make sure that all tissue is completely pulverized. [A commercial floor-model paintshaker works great, too].
4. Spin the plates briefly (30 sec at 3000 rpm at 4ºC) to bring the powdered tissue down. The samples are going to start to thaw at this point, which is OK. Freeze the samples again at -80ºC if not using immediately.
5. Uncap the tubes (one strip at a time) and add ~250 ul CTAB buffer per well using an 8-channel pipet (3 x 85ul is OK). Recap the tubes tightly, cover the plates with inverted lids and shake the plates vigorously to dislodge the metal beads. At this point, make sure that all of the samples are completely thawed and the CTAB in the wells is NOT frozen/precipitated (a short incubation at RT or 37ºC should help). Spin the plates briefly (30 sec at 3000 rpm at 4ºC or RT) to bring the samples down.
6. Uncap the strips (throw the caps away) and cover the plates with the lid. Incubate the plates at 65ºC for 30 minutes.
7. Cool the samples to RT (placing the plates on ice or at 4ºC speeds up the process). Add 250ul chloroform (or a 24:1 Chl:IAA mix) per well using an 8-channel pipet (3 x 85ul is OK). Using an 8-channel pipet and wide-bore tips (Fisher 21-197-2B), mix the samples by pipeting them up and down (set the pipet volume to no more than 60ul to avoid sample cross-contamination by drops jumping onto the pipet).
8. Spin the samples for 15min at 3000 rpm at 4ºC or RT. In a meanwhile, prepare new 96-well plates with 200ul isopropanol per well (no need to cap the strips if using immediately, but a lid will help to keep the smell away)
9. Transfer 200ul of each sample (aqueous phase) into the plate with isopropanol using an 8-channel pipet, one strip at a time. Be careful not to transfer cell debris from the interface.
10. As you are transferring, mix the samples by pipeting using an 8-channel pipet (set the pipet volume to no more than 60ul to avoid sample cross-contamination by drops jumping onto the pipet).
11. Spin the samples for 30min at 3000 rpm at 4ºC.
12. Discard the supernatant by inverting the strips (one at a time) and let the samples sit up-side-down on a paper towel for ~1min to remove as much isopropanol as possible. Be careful not to loose the DNA pellets.
13. To wash the pellets, place the strips right-side-up in a rack and add ~250ul of 70% EtOH with an 8-channel pipet (3x 85ul is OK).
14. Spin the samples for 15-30min at 3000 rpm at 4ºC (longer time ensures pellets sticking better to the bottom of the tubes).
15. Discard the wash by inverting the strips (one at a time) and let the samples sit up-side-down on a paper towel for ~1min to remove as much EtOH as possible. Be careful not to loose the DNA pellets. Place the strips back into the rack right-side-up and air-dry the DNA pellets in the flow hood for at least one hour. Make sure no EtOH remains in the wells and the pellets are completely dry.
16. Resuspend the DNA by adding 200-400ul of deionized water. Cover with the lid (no cap strips are necessary). Vortex the plate several times and let it sit for 1h-overnight at 4ºC for the pellets to dissolve.
17. Vortex again when ready to use and spin for >1min at 3000 rpm at 4ºC to pellet any insoluble material. Use 1ul DNA per 10ul PCR reaction. Freeze the plate at -20ºC if not using in the next few days (no need to cap the strips, an overall lid will do the job for up to 6 months, just don’t invert the samples). 4ºC is OK for short-term storage (up to one month).
20mM EDTA, pH8
100mM Tris-HCl, pH8
3% CTAB (cetyltrimethylammonium bromide, Calbiochem, #219374, 100g)
(optional) 1% beta-mercaptoethanol (add fresh before use)
Daisy BB 4.5mm steel zink-plated airgun shot (available at WalMart at <$5 for 6000 beads!)
Plates (racks of 12 microtube strips of 8):
ISC BioExpress www.bioexpress.com P-8705-2 $32.90 per pack of 10 racks
or Genesee Scientific www.geneseesci.com 14-361 $34.65 or less per pack of 10 racks
Replacement 8-tube strips:
ISC BioExpress P-8705-1 $19.90 per pack of 120 strips
or Genesee Scientific 14-360 $21.00 or less per pack of 125 strips
Replacement 8-cap strips:
ISC BioExpress P-8705-C $9.70 per pack of 120 strips (also compatible with the ISC BioExpress tubes) or Genesee Scientific 14-150 $18.00 or less per pack of 125 strips