4. Aspiration system: in-line or bioreagent vacuum pump and
tubing (see Notes 10 and 11).
5. Glass beads for cell culture plating, cleaned and autoclaved.
6. 15 mL conical tubes.
2.3 Isolating Clones
in Solid Media
and Long-Term
Storage
1. Sterile mixed cellulose ester syringe filters, 0.22 μm pore size,
33 mm diameter (see Note 12).
2. 5 mL syringes without needle attached.
3. Glycerol solution: 60% glycerol in ultrapure water, autoclaved.
4. À80
C and/or À160
C ultralow temperature freezer(s).
5. 15 mL conical tubes.
2.4 Nested PCR
Using a Semi-random
Primer to Determine
Transposon
Insertion Site
1. Genomic DNA purification kit.
2. UV/vis spectrophotometer.
3. Standard PCR reagents.
4. Primers (Table 1).
5. Thermal cycler.
6. 10Â Tris/borate/EDTA (TBE) running buffer solution:
0.89 M Tris base, 0.89 M boric acid, 20 mM EDTA in distilled
water, pH 8.3.
7. Agarose gel: 0.8% agarose in 1Â TBE solution.
8. 6Â DNA loading dye solution: 40% sucrose, 0.25% xylene
cyanol FF, 0.25% bromophenol blue in distilled water.
9. Nucleic acid staining solution.
10. Electrophoresis power supply.
11. DNA molecular weight marker.
12. Nucleic acid gel documentation system.
13. Capillary DNA sequencer or DNA sequencing outsourced to a
commercial sequencing company.
3 Methods
All procedures should be performed within a biosafety cabinet type
2A to reduce risk of contamination of transconjugants and for
biosafety. The random transposon mutagenesis protocol and conjugation plasmids were originally described and generously donated
by Dr. Bourhy and Dr. Picardeau, respectively [16, 17, 41].
80
Christopher J. Pappas et al.
tubing (see Notes 10 and 11).
5. Glass beads for cell culture plating, cleaned and autoclaved.
6. 15 mL conical tubes.
2.3 Isolating Clones
in Solid Media
and Long-Term
Storage
1. Sterile mixed cellulose ester syringe filters, 0.22 μm pore size,
33 mm diameter (see Note 12).
2. 5 mL syringes without needle attached.
3. Glycerol solution: 60% glycerol in ultrapure water, autoclaved.
4. À80
C and/or À160
C ultralow temperature freezer(s).
5. 15 mL conical tubes.
2.4 Nested PCR
Using a Semi-random
Primer to Determine
Transposon
Insertion Site
1. Genomic DNA purification kit.
2. UV/vis spectrophotometer.
3. Standard PCR reagents.
4. Primers (Table 1).
5. Thermal cycler.
6. 10Â Tris/borate/EDTA (TBE) running buffer solution:
0.89 M Tris base, 0.89 M boric acid, 20 mM EDTA in distilled
water, pH 8.3.
7. Agarose gel: 0.8% agarose in 1Â TBE solution.
8. 6Â DNA loading dye solution: 40% sucrose, 0.25% xylene
cyanol FF, 0.25% bromophenol blue in distilled water.
9. Nucleic acid staining solution.
10. Electrophoresis power supply.
11. DNA molecular weight marker.
12. Nucleic acid gel documentation system.
13. Capillary DNA sequencer or DNA sequencing outsourced to a
commercial sequencing company.
3 Methods
All procedures should be performed within a biosafety cabinet type
2A to reduce risk of contamination of transconjugants and for
biosafety. The random transposon mutagenesis protocol and conjugation plasmids were originally described and generously donated
by Dr. Bourhy and Dr. Picardeau, respectively [16, 17, 41].
80
Christopher J. Pappas et al.