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

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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