SINGLE FLY DNA PREPS FOR PCR
Gloor et al., (1993)Genetics135:81-95
A. DNA PREPARATION PROTOCOL:
1) Squishing buffer (SB) is 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl.
Use 50 l per fly; add 1 l Proteinase K from frozen stock (10 mg/ml).
2) Place one fly in a 0.5 ml tube and mash the fly with a pipette tip containing 50 l of
SB, but no not expel any liquid until after squishing.
3) Incubate at 25-37 C (or room temp.) for 20-30 minutes. This and step 3 are
conveniently done in a thermal cycler.
4) Inactivate the Proteinase K by heating to 95 C for 1-2 minutes.
This preparation can be stored at 4 C for months. Typically use 2 l of the DNA prep in
a 20 l reaction volume. It does not matter if fly parts (wings, bristles, legs) are
inadvertently added to the PCR mixture.
Procedure can also be done in a 96-well microtiter plate.
Can also be done with fly embryos. Use 20 uL of SB + 0.4 uL ProtK (10mg/mL)