Biology Reference
In-Depth Information
DNA free of contaminants (
Moreira 1998
). The agarose-embedded DNA can be
used directly because the PCR is unaffected by the presence of high-quality low-
melting-point agarose in concentrations up to 0.3%.
The use of degraded and fragmented DNA as a template for the PCR gener-
ally should be avoided because it reduces the efficiency of the PCR and limits the
size of the products that can be amplified (
Golenberg et al. 1996
).
Concentrations of 0.05-1.0
μ
g of genomic DNA typically are used to amplify
single-copy genes with the standard PCR. Less DNA (0.5-2 ng) can be used to
amplify multiple-copy genes such as nuclear ribosomal RNA genes because these
genes are repeated
≈
200-500 times in the eukaryotic genome. It even may be
possible to conduct PCR on ancient or degraded DNA if the template is “recon-
structed” (
Golenberg et al. 1996
).
Table 8.5
describes a standard protocol for preparing genomic DNA suitable
for the PCR from a single
D. melanogaster
(
Jowett 1986
). Other techniques are
possible.
Table 8.5: Extracting Genomic DNA From a Single
Drosophila melanogaster.
1.
In a 1.5-ml microfuge tube, freeze a fly in liquid nitrogen. (Store at
−
70 °C until needed.)
2.
Thaw and add 100
μ
l of 10 mM Tris•HCl (pH 7.5) 60 mM NaCl, 50 mM EDTA, 0.15 mM spermine, and
0.15 mM spermidine.
3.
Grind fly with a yellow pipet tip.
4.
Add 100
μ
l of 1.25% SDS, 0.3 M Tris•HCl, 0.1 M EDTA, 5% sucrose, and 0.75% freshly added
diethylpyrocarbonate (DEP).
5.
Mix and incubate 30-40 min at 60 °C to ensure lysis of the nuclei.
6.
Cool and add 30
μ
l of 8 M potassium acetate.
7.
Cool for 45 minutes on ice.
8.
Spin for 1 minutes in a microfuge.
9.
Remove supernatant, avoiding the lipid on the surface and add 2 volumes of ethanol.
10.
Leave at room temperature.
11.
Spin for 5 minutes and pour off the supernatant.
12.
Wash the pellet with 70% EtOH.
13.
Dry under vacuum.
14.
Take up pellet in 25
μ
l of TE buffer (10 mM Tris and 1 mM EDTA).
The method lyses the nuclei once the tissue is broken up. The SDS and protein form complexes. DEP
is a protein denaturant and nuclease inhibitor. The protein/SDS complexes are precipitated by adding
potassium, leaving the DNA in solution. The final DNA is contaminated with RNA, which can be removed by
adding RNase to a concentration of 100
μ
g/ml.
(Adapted from Jowett 1986.)
