10. Petroff-Hausser chamber.
11. Female Golden Syrian hamsters, 3- to 4-weeks-old.
12. 1 ml insulin syringes U-100 with 26 G Â ½
00 needle.
13. 3 ml syringes with 25 G Â 5/8
00 needle.
14. 5 ml EDTA blood collection tubes.
15. Precision balance (to weigh animals).
16. Isoflurane and isoflurane vaporizer.
17. Scalpels.
18. Scissors.
19. Forceps.
20. 1.5 ml microcentrifuge tubes.
21. 1 ml cryotubes.
22. Phosphate buffered saline (PBS): 1.8 mM KH 2 PO 4 ,137 mM
NaCl, 10 mM Na 2 HPO 4 , 2.7 mM KCl, pH 7.2. Autoclave and
then store at room temperature (RT).
23. Tissue homogenizer (Omni Bead Ruptor).
2.2 Preparation
of Genomic Libraries
1. DNA extraction kit.
2. Sonicator with cup horn.
3. Tris-acetate-EDTA (TAE) buffer (50Â): 2 M Tris base, 1 M
acetic acid, and 50 mM disodium EDTA.
4. Ethidium bromide: 10 mg/ml solution in water.
5. Agarose gel: 2% gel in 1Â TAE buffer with 0.5 μg/ml of
ethidium bromide (5 μl of 10 mg/ml ethidium bromide in
100 ml gel).
6. Microvolume spectrophotometer.
7. dCTP + ddCTP: 9.5 mM dCTP, 0.5 mM ddCTP solution in
water.
8. Terminal deoxynucleotidyl transferase (TdT): 30 U/μl.
9. Thermal cycler.
10. Pfu DNA polymerase and master mix.
11. PCR purification kit.
12. Oligonucleotides (Tables 1 and 2).
13. Fluorometer.
3 Methods
3.1 Assembly
of the Pool of Mutants
(Input Pool)
1. Grow each transposon insertion mutant individually in EMJH
supplemented with 50 μg/ml kanamycin at 30
C under
100 rpm agitation to a density of 10
8 leptospires/ml.
Transposon Sequencing in Leptospira interrogans
99
11. Female Golden Syrian hamsters, 3- to 4-weeks-old.
12. 1 ml insulin syringes U-100 with 26 G Â ½
00 needle.
13. 3 ml syringes with 25 G Â 5/8
00 needle.
14. 5 ml EDTA blood collection tubes.
15. Precision balance (to weigh animals).
16. Isoflurane and isoflurane vaporizer.
17. Scalpels.
18. Scissors.
19. Forceps.
20. 1.5 ml microcentrifuge tubes.
21. 1 ml cryotubes.
22. Phosphate buffered saline (PBS): 1.8 mM KH 2 PO 4 ,137 mM
NaCl, 10 mM Na 2 HPO 4 , 2.7 mM KCl, pH 7.2. Autoclave and
then store at room temperature (RT).
23. Tissue homogenizer (Omni Bead Ruptor).
2.2 Preparation
of Genomic Libraries
1. DNA extraction kit.
2. Sonicator with cup horn.
3. Tris-acetate-EDTA (TAE) buffer (50Â): 2 M Tris base, 1 M
acetic acid, and 50 mM disodium EDTA.
4. Ethidium bromide: 10 mg/ml solution in water.
5. Agarose gel: 2% gel in 1Â TAE buffer with 0.5 μg/ml of
ethidium bromide (5 μl of 10 mg/ml ethidium bromide in
100 ml gel).
6. Microvolume spectrophotometer.
7. dCTP + ddCTP: 9.5 mM dCTP, 0.5 mM ddCTP solution in
water.
8. Terminal deoxynucleotidyl transferase (TdT): 30 U/μl.
9. Thermal cycler.
10. Pfu DNA polymerase and master mix.
11. PCR purification kit.
12. Oligonucleotides (Tables 1 and 2).
13. Fluorometer.
3 Methods
3.1 Assembly
of the Pool of Mutants
(Input Pool)
1. Grow each transposon insertion mutant individually in EMJH
supplemented with 50 μg/ml kanamycin at 30
C under
100 rpm agitation to a density of 10
8 leptospires/ml.
Transposon Sequencing in Leptospira interrogans
99