Figure 1
- Isolation of DNA from Lepstospira and Mycobacterium with
E6870.
A, Leptospira tarasovi (108 cells) was sedimented at 14,000 g
for 20 min. The sedimented cells were resuspended in 50 mM Tris, pH 8.0, 50 mM EDTA, 100
mM NaCl, 1% SDS and 120 U of proteinase K or 100-400 U of E6870 and the mixture was
incubated at 50oC for 2 h. The solution was then centrifuged at 10,000 g
for 10 min and the supernatant deproteinized by phenol extraction
(phenol:chloroform:isoamylalcohol = 25:24:1). Before precipitation, 1 µg tRNA was added
as carrier. After ethanol precipitation and washing with 70% ethanol the dried pellet was
dissolved in 20 µl TE (Tris-EDTA) and 10 µl of this solution was electrophoresed on a
1.5% agarose gel along with molecular weight markers. Lanes: 1, l-HindIII; 2, 120 U
proteinase K; 3, 100 U; 4, 200 U; 5, 300 U and 6, 400 U of E6870.
B, Mycobacterium bovis previously inoculated into milk was isolated
as follows: a milk sample (1 ml) containing 108 CFU/ml of M. bovis was
incubated overnight with 720 U proteinase K (Boehringer, Mannheim, Germany), 360 U (lane
3), or 720 U (lane 4) of E6870 at 56oC in buffer 10 mM Tris, 5 mM EDTA, pH 8.0,
1.5% SDS. The suspension was acidified with 200 µl 10% acetic acid and proteins were
removed with 1 ml of a mixture containing phenol (3x) as shown in Figure 1A. The remaining
procedure was as described in 1A. An aliquot (20% of total volume) was electrophoresed on
agarose gel (1.5%) followed by ethidium bromide staining. Lanes: 1, l-HindIII; 2,
720 U proteinase K; 3, 360 U; 4, 720 U E6870; 5, no proteinase added.
Figure 2
- PCR amplification of DNA from Leptospira and Mycobacterium.
A, DNA (10 ng) from Leptospira was subjected to 30 amplification cycles
consisting of denaturation at 94oC for 90 s, annealing at 58oC for
90 s and extension at 72oC for 2 min. The last extension step lasted 10 min.
The other components of the mix were: 2 mM MgCl2, 0.2 mM dNTP, 50 mM KCl, 10 mM
Tris-HCl, pH 8.0, 0.1% Triton X-100, 10 U Taq polymerase and 800 nM each of primers
Lep13/Lep14 (9). Lane 1, 100 bp; lane 2, negative control; lanes 3-8 are L. bratislava,
L. hardjo, L. norma, L. hardjobovis, L. mini, and L.
neguita, respectively.
B, Mycobacterial DNA (0.001-500 ng) was subjected to 42 cycles of
amplification consisting of denaturation at 95oC for 30 s, annealing at 68oC
for 60 s and extension at 72oC for 30 s. The final extension step was carried
out at 72oC for 30 min. The other components of the mixture were: 50 mM KCl, 10
mM Tris, pH 8.3, 2.0 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and 0.4 mM dUTP
or dTTP, 0.5 µmol each of primers BW6/BW7 (10,12), 0.5 U of uracil DNA glycosylase (Gibco
BRL, Gaithersburg, MD), and 2.5 U Taq DNA polymerase. Before PCR the mixture was
preincubated at 37oC for 10 min. The glycosylase was then heat inactivated by
incubation at 95oC for 10 min. Lane 1, 100 bp; lanes 2-7 are DNA from cow's
milk collected from different animals.
