SINGLE-FLY DNA PREPS FOR PCR
Gloor et al. 1993 Genetics 135:81-95
We have developed a simple method for the rapid and reproducible isolation of DNA from single flies for amplification by the polymerase chain reaction (PCR) (Saiki et al, Science 239: 487), and direct sequencing by asymmetric PCR (Gyllensten and Erlich, Proc. Nat. Acad. Sci. 85: 7652). The simplicity of this procedure means that the problem of contamination with other amplified or cloned DNA is greatly reduced. Sufficient DNA is obtained from one fly for a minimum of 50 PCR analyses, and the DNA is stable for at least one month in the refrigerator. A simple modification of this technique allows the isolation of DNA suitable for use in inverse PCR (Ochman et al, Genetics 120: 621-623). These methods substantially reduce the time involved in DNA isolation, and among other uses, allows the PCR to be used to monitor the segregation of an allele for which there is no phenotype or transposition of an unmarked P element (Engels et al. Cell 62: 515-525).
A. DNA PREPARATION PROTOCOL:
1. The squishing buffer (SB) is 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, and 200ug/ml Proteinase K, with the enzyme diluted fresh from a frozen stock each day (to 1ml of SB add 10ul of 10mg/ml Proteinase K stock).
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 25-37 C (or room temp.) for 20-30 minutes.
4. Inactivate the Proteinase K by heating to 95 C for 1-2 minutes.
NOTES: This preparation can be stored at 4 C for months. We typically use 1 ul of the
DNA prep in a 10-15 ul reaction volume. It does not matter if fly parts (wings, bristles,
legs) are inadvertantly added to the PCR mixture. Product will typically start to appear
after 24-25 cycles, but 28-30 cycles seems to give maximal yield (35 cycles OK). Increasing the number of flies does not seem to increase the signal significantly, probably due to increasingconcentrations of inhibitors. There should be no problem scaling up the number of fliesscreened if the volume is increased proportionately. 85 C heat inactivation allows longerfragments to be amplified -- up to more than 9 kb with ³long PCR².
Embryo Note:
For Embryos it is best if you use between 5-10 embryos and only use 20ul of SB buffer. Also, for squishing embryos it is best done in the cap of a microcentrifuge tube with 2-5ul of SB and the tip of a metal pointer. After the embryos are squished then just spin embryo/SB solution to bottom of tube. It is also a good idea to use at least 2ul of DNA prep.
PCR Protocol
- Preparation of Master Mix for Reaction
Solutions needed: 10X PCR Buffer, 2mM dNTP’s, 200pM of each Primer, Taq DNA Polymerase, and Template from above.
- For 10 rxn’s Master Mix use the followingFor 40 rxn’sFor 1 rxn
159ul of H20639ul of H2013 ul H2O
20ul of 10X Reaction Buffer (-20 in PCR Box) 80ul of 10X Buffer 2 ul Buffer
4ul of dNTP’s [2mM] 16ul of dNTP’s 1 ul dNTP’s
3ul of primer #1 [10μM] 12ul of primer #1 1 ul primer #1
3ul of primer #2 [10μ M] 12ul of primer #2 1 ul primer#2
1ul of Taq DNA Polymerase (-20 in PCR Box) 1ul of Taq Polymerase 1 ul Taq
190ul Total760ul Total19 ul Total
to a PCR tube add 19ul of Master Mix and 1ul (Fly) or 2ul (Embryo) of Template
- PCR Cycle (Cycle 25-35)
94C for 5 min
94C for 30 sec
60C for 30 sec
72C for 1 min (or 1 min/ kb of sequence)
70C for 7 min
4C ------