User:Robert J. Schiemann/Notebook/Lipsick Lab/Protocols/Fly PCR

Single-fly DNA prep protocol for PCR

1. The squishing buffer (SB) is 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, and 200 ug/ml Proteinase K, with the enzyme diluted fresh from a frozen stock each day. -- add Proteinase K at 1:50 dilution from stock just before use

2. Place one fly in a 0.5 ml tube and mash the fly for 5 - 10 seconds with a pipette tip containing 50 ul of SB, without expelling any liquid (sufficient liquid escapes from the tip). Then expel the remaining SB.

3. Incubate at 25-37o C (or room temp.) for 20-30 minutes. -- I use 37o C water bath for 25 minutes.

4. Add 1ul of 0.1M PMSF (phenylmethylsulfonylfluoride), then heat to 65oC for 10 - 15 minutes to denature any proteins not inactivated by the proteinase.

PCR reaction for neoFRT and flpase primers (uses double MgCl2 concentration)

DNA 				1 ul 10x PCR buffer		       2 ul 10 mM dNTPs			0.4 ul 50 mM MgCl2			1.2 ul 10 uM for. primer		1 ul 10 uM rev. primer		1 ul ddH2O				13.2 ul Taq (Invitrogen)		0.2 ul 				20 ul