A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 68

2.2.2. Plasmids (see Note 3, GenBank Accession Numbers in Bold)
1. pTABhFlgB-Kan (source of kanamycin selectable cassette) (EF488744) .
2. pTABhFlaB-Gent (source of gentamicin selectable cassette) (EF488745) .
3. pBhSV-2 ( B. hermsii shuttle vector) (EF488742) .
4. pOK12 (plasmid backbone for construction of inactivation and reconstitution
plasmids) (11) (EF488748) .
5. pOKvtp (plasmid containing the 5193-bp vtp locus, kan-r) (EF488743) .
6. pOKvtpKO ( vtp inactivation plasmid, kan-r) (EF488747) .
7. pOKvtpRECON ( vtp reconstitution plasmid, gent-r) (EF488746) .
2.2.3. Primers (Listed 5´-3´, Restriction Endonuclease Tags Are
Underlined, Invitrogen, Carlsbad, CA)
1. 5´ BhflgB+NgoMIV AT GCCGGC GTTAAAGAAAATTGAAATAAACTTG.
2. 3´ BhflgB+NdeI ATT CATATG AACACCCTCTATATCAC.
3. 5´ BhflaB+NgoMIV TA GCCGGC AATTCCTAATCAGAAAAATGTG.
4. 3´ BhflaB+NdeI T CATATG TCATTTCCTCCGTG.
5. VTP F2+AvrII AAT CCTAGG GCAAATACTATGCTCTTGATGG.
6. VTP B3+AscI ATT GGCGCGCC TCTTTAATGCCTTATCAC.
7. pOK12F1+AscI T GGCGCGCC TCGCCCTTCCCAACAGTTG.
8. pOK12B1+AvrII A CCTAGG GCGTATTGGAGCTTTCGCG.
9. vtpF1+ScaI AA AGTACT TAACCGCTGCTAAAGATGCAGTAG.
10. vtpB1+SpeI AA ACTAGT CACCCTTTTTAAGCTCTGGGC.
11. ReconF+XmaI GTG CCCGGG GATTATAAGATTTAACAC.
12. ReconR+SpeI TT ACTAGT AATATCCTAAGTCGATGAACAG.
13. Gent5´+XmaI ATTCG CCCGGG CCGGCAATTCCTAATCAGAAAAATGTGG.
14. Gent3´+SpeI AA ACTAGT CTCGGCTTGAACGAATTGTTAGG.
15. BhOriF+NotI AAT GCGGCCGC TAAGTCCAAAAGCGTCCC.
16. BhOriR+SphI ATT GCATGC TAAAATCTTCTTGCCCGC.
17. LONG VTP F1 AGAGAGATATTTAGAATTAAAAGAAAAGC.
18. LONG VTP B56 GTCAAGAAGAGCATTTGTCACAC.
2.3. Nucleic Acid Cloning Materials
1. GeneAmp PCR Core Kit (Roche Molecular Systems, Branchburg, NJ).
2. Expand Long Template PCR System Kit (Roche Diagnostics, Indianapolis, IN)
( see Note 4 ).
3. TOPO TA Cloning Kit (Invitrogen).
4. QIAquick PCR Purification Kit and the QIAquick Gel Extraction Kit (Qiagen,
Valencia, CA).
5. QIAprep Spin and Plasmid Maxi Kits (Qiagen).
6. Restriction enzymes and T4 DNA ligase (New England Biolabs, Beverly, MA).
7. BlueView Nucleic Acid Stain (Sigma-Aldrich) ( see Note 5 ).
8. QIAtissue Kit (Qiagen).