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 increasing concentrations of inhibitors. There should be no problem scaling up the
number of flies screened if the volume is increased proportionately. 85 C heat
inactivation allows longer fragments 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 25ul 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
1. 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.
2.
For 10 rxn’s Master Mix use the following
For 40 rxn’s
159ul of H20
20ul of 10X Reaction Buffer (-20 in PCR Box)
4ul of dNTP’s [2mM]
3ul of primer #1 [10μM]
3ul of primer #2 [10μ M]
1ul of Taq DNA Polymerase (-20 in PCR Box)
190ul Total
639ul of H20
13 ul H2O
80ul of 10X Buffer
2 ul Buffer
16ul of dNTP’s
1 ul dNTP’s
12ul of primer #1
1 ul primer #1
12ul of primer #2
1 ul primer#2
1ul of Taq Polymerase 1 ul Taq
760ul Total
19 ul Total
For 1 rxn
to a PCR tube add 19ul of Master Mix and 1ul (Fly) or 2ul (Embryo) of Template
3. 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 ----------