Single Fly Genomic DNA Prep

Published on Author Giorgio Gilestro

From Georg Dietzl in Barry Dickson’s Lab, IMP Vienna 12/2002
SQUISHING BUFFER (SB)

  • 10 mM Tris-HCl pH8
  • 1 mM EDTA
  • 25 mM NaCl
  • 200 g/ml Proteinase K

Procedure

  1. 200 g/ml Proteinase K are added to the squishing buffer (SB) freshly from 20X stock every day.
  2. Place 1-2 (I always take 2) flies in a 0.5 ml tube (I use 96 microwell plates from MJ Research since I usually do a larger number of preps at once). Mash the flies for 5-10 sec with a yellow pipette tip containing 50 l of SB, without expelling any liquid (sufficient liquid escapes from the tip). Then expel the remaining SB. (If you use 96 microwell plates you can use a 100 or 300 l 8-channel pipette for mashing the flies. This works really well and is much faster for a larger number of samples)
  3. Incubate at 37 o C for 30 min
  4. Inactivate the Proteinase K by heating to 95 o C for 3 min.
  5. Spin down the solid junk briefly and pipette the supernatant to a new tube / microwell plate.
  6. That’s it

96 Well modifications (GG)

The protocol can be executed directly on 96 wells plates.

  1. Place one fly per well
  2. Add few microliters of SB then spin down using a centrifuge with plates adapters
  3. Squeeze flies using either a multi well pipette with yellow tips or the custom made 3D printed squeezing tool

Vosshall Lab Modifications and Notes:
(1) Use Disposable Kontes Pellet Pestle for grinding. These come with RNase/DNase free 1.5 ml microcentrifuge tubes that we use to freeze the 2 flies. Packaged separately but included in the catalog number below are RNase/DNase free disposable pestle grinders that are very effective at grinding flies.
Fisher Scientific Catalog Number: K749520-0090 2003 list price: $88.30/100 tubes + pestles
(2) Make a 10mg/ml Roche Proteinase K stock solution in 1XPBS and freeze in small aliquots at -20 o C. Immediately before use, add 20ul of this Proteinase K stock solution to 1 ml of SB
(3) Use 1 ul of this crude DNA prep for PCR reactions.

Link to PDF: SingleFlyGenomic