wrengels at facstaff.wisc.edu
Thu Nov 23 21:31:09 EST 1995
In article <MAILQUEUE-101.951124130924.352 at sbsnov1.auckland.ac.nz>,
g.warman at AUCKLAND.AC.NZ wrote:
> There is a reference to Gloor and Engels single fly DNA preps in the
> Drosophila information newsletter volume 1 (January). I do not have
> access to this newsletter and would really like a copy of the
The note from DIN volume 1 is appended. We still use approximately the
same procedure except that the heat treatment is now done at 85[o] instead
of 95[o] when proteinase K is used. This allows longer fragments to be
amplified -- up to more than 9 kb with ³long PCR².
The same protocol as below is also given in Gloor et al., (1993, Genetics
135:81-95), which might be better to cite, since Genetics is more widely
23 November 1995
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William R. Engels : WREngels at facstaff.wisc.edu
Genetics Department, 445 Henry Mall : office: (608) 263-2213
University of Wisconsin : lab: (608) 262-5578
Madison, WI 53706 : FAX: (608) 262-2976
SINGLE-FLY DNA PREPS FOR PCR
Greg Gloor and William Engels, Dept. of Genetics, Univ. of Wisconsin,
Madison, WI 53706, 608-263-2213, FAX/262-2976, WRENGELS at facstaff.wisc.edu
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 200 ug/ml Proteinase K, with the enzyme diluted fresh from a
frozen stock each day.
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[o]C (or room temp.) for 20-30 minutes.
4. Inactivate the Proteinase K by heating to 95[o]C for 1-2 minutes.
NOTES: This preparation can be stored at 4[o]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. 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
B. DNA PREPS FROM MANY INDIVIDUAL FLIES
A similar method can be used with 96-well (8 x 12) micro plates to prepare
DNA from a large number of individual flies. Up to 80 flies can be tested
with a single plate.
1. Place one anesthetized or frozen fly in each well. All rows (A-H) can
be utilized, but leave the first and last columns (1 and 12) empty to
ensure complete heating in step 3.
2. Add 50 ul of SB to each well and macerate each fly with a toothpick for
5-10 seconds. Then cover the plate with an adhesive-backed strip to
prevent evaporation and contamination. Incubate at room temperature as
3. Use two standard-size heating blocks (9.5 x 7.5 cm) pre-heated to
95[o]C to inactivate the Proteinase K. Sandwich the micro plate, with its
adhesive lid still in place, between the two heating blocks. The lower
block should be inverted so that the tube holes are facing downward and
the flat surface is touching the bottom of the micro plate. After 2-3
minutes remove the micro plate and set it on the bench top with the upper
hot block still on top. This upper block will gradually cool to room
temperature, preventing condensation on the underside of the adhesive.
NOTES: It is helpful to tape a piece of waxed paper over the open micro
plate while the PCR tubes are being set up. That way the DNA samples can
be drawn from each well by poking the pipet tip through the waxed paper.
This procedure reduces the possibility of contamination and helps to keep
track of which wells have been used.
C. INVERSE PCR
PMSF (phenylmethylsulfonylfluoride) can be used instead of the 95[o]C
treatment to inactivate the Proteinase K if the DNA preps are to be used
for inverse PCR or other methods that require double-stranded DNA.
1. Add 1 ul of 0.1 M PMSF to the fly prep following step 3 of protocol A
above. Then heat the mixture to 65[o]C for 10 - 15 minutes to denature any
proteins not inactivated by the proteinase.
2. Add 4 ul of fly supernatant to 16 ul of 1.25X NdeII buffer (125 mM
Tris-HCl pH 7.6, 12.5 mM MgCl, 188 mM NaCl, 1.25 mM DTT). Add 0.5 ul of
NdeII (BRL) and incubate at room temp. for 15 min. Inactivate the enzyme
by heating to 65[o]C for 15 min.
3. Take 3 ul of digested DNA and add to 7 ul of ligation mix (5 mM MgCl2,
20 mM DTT, 0.8 mM ATP). Add 0.5 ul T4 DNA ligase (NEB) and incubate at
room temperature for 20-30 min. Inactivate the enzyme by heating to 95[o]C
for 2-3 min; this also serves to nick the DNA.
NOTES: For the first attempt with a new insertion, we recommend using the
following conditions: denature at 94[o]C for 45 sec., anneal at 60[o]C for
45 sec., extend at 72[o]C for 4 min; try 30 - 35 cycles. - The restriction
enzyme appears to be the most critical component in this protocol. The
enzyme must be specific under conditions of very low DNA concentration.
Sau3A1, for example, is too promiscuous and digests at several sites in
addition to its canonical restriction sequence. The protocol should work
with other enzymes. The protocol has been used for a combined
inverse/asymmetric PCR procedure to get DNA sequences flanking P element
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