3. Incubate the tubes for 5–28 days (growth rate is dependent
upon which gene has been disrupted) at 30
C with or without
agitation until Leptospira growth is observed as turbid media.
4. Once samples become turbid, distribute a 10 μL aliquot onto a
microscope slide to confirm Leptospira culture is not contaminated by dark-field microscopy (see Note 36).
5. If not contaminated and growth is robust and at mid- to lateexponential growth, sample can be labeled (e.g., Tn1, Tn2,
etc.) and used for downstream applications and long-term
storage at À80
C or À160
C (see Notes 37–39).
3.5 Nested PCR
Using a Semi-random
Primer to Determine
Transposon
Insertion Site
To determine transposon inserted site, nested PCR using a semirandom primer is completed as previously described [18, 42].
1. Purify genomic DNA from 1–2 mL of non-contaminated Tn
Leptospira sample (see Note 40).
2. Optional: Use purified genomic DNA to confirm the sample is
the correct Leptospira species and/or harboring the transposon
by running PCR reactions for a species-specific gene (e.g.,
LEPBIa0024F and R for L. biflexa) or PCR for a transposonspecific primer set (e.g., VTK1-TKS2 and VTK2-TKS2)
(Table 1).
3. Set up a mix for round 1 of nested PCR by using semi-random
primers with the following reagents and quantities per reaction
tube (total volume ¼ 24.55 μL): 2.5 μL of 10Â Taq polymerase
Fig. 6 EMJH solid medium plates with transconjugants. Left: Plate without 1% rabbit serum showing
transconjugants with eight colony stabs (e.g., arrows). Right: Plate enriched with 1% rabbit serum showing
transconjugants of various motilities. Care must be taken to ensure colony stabs are clonal; plating on more
than three EMJH plates is recommended when high transformation efficiency/high density is anticipated.
Noble agar concentration for both plates is 1%
86
Christopher J. Pappas et al.
upon which gene has been disrupted) at 30
C with or without
agitation until Leptospira growth is observed as turbid media.
4. Once samples become turbid, distribute a 10 μL aliquot onto a
microscope slide to confirm Leptospira culture is not contaminated by dark-field microscopy (see Note 36).
5. If not contaminated and growth is robust and at mid- to lateexponential growth, sample can be labeled (e.g., Tn1, Tn2,
etc.) and used for downstream applications and long-term
storage at À80
C or À160
C (see Notes 37–39).
3.5 Nested PCR
Using a Semi-random
Primer to Determine
Transposon
Insertion Site
To determine transposon inserted site, nested PCR using a semirandom primer is completed as previously described [18, 42].
1. Purify genomic DNA from 1–2 mL of non-contaminated Tn
Leptospira sample (see Note 40).
2. Optional: Use purified genomic DNA to confirm the sample is
the correct Leptospira species and/or harboring the transposon
by running PCR reactions for a species-specific gene (e.g.,
LEPBIa0024F and R for L. biflexa) or PCR for a transposonspecific primer set (e.g., VTK1-TKS2 and VTK2-TKS2)
(Table 1).
3. Set up a mix for round 1 of nested PCR by using semi-random
primers with the following reagents and quantities per reaction
tube (total volume ¼ 24.55 μL): 2.5 μL of 10Â Taq polymerase
Fig. 6 EMJH solid medium plates with transconjugants. Left: Plate without 1% rabbit serum showing
transconjugants with eight colony stabs (e.g., arrows). Right: Plate enriched with 1% rabbit serum showing
transconjugants of various motilities. Care must be taken to ensure colony stabs are clonal; plating on more
than three EMJH plates is recommended when high transformation efficiency/high density is anticipated.
Noble agar concentration for both plates is 1%
86
Christopher J. Pappas et al.