DNA isolated from the St. Maries and Virginia strains was digested with XbaI and HindIII restriction enzymes (New England Biolabs) and separated on a 0.7% agarose gel. Gel slices corresponding to the appropriate size of the aaap locus were extracted using the QIAquick Gel Extraction kit (Qiagen). The DNA was ligated with XbaI-HindIII-digested pBluescript II KS- (Stratagene) vector. Transformed colonies, grown in E. coli TOP10 or Stbl2 (Invitrogen) cells, were screened for inserts containing the aaap locus by membrane hybridization using the Digoxigenin-labeled aaap probe. Membrane hybridization was carried out as directed (Roche Applied Science) with denaturation in 0.5 M NaOH, 1.5 M NaCl for 15 min, neutralization in 0.5 M Tris [pH 7.5], 1.5 M NaCl for 15 min, followed by 10 min in 2× SSC. Hybridization was as described for Southern blots. Cloning of the St. Maries aaap locus from DNA isolated from infected ISE6 cells was performed via Polymerase Chain Reaction (PCR) using primers 5′-CAG GCC CAA AAT CGC GTC ATC C-3′ and 5′-CCC TAG CCC TAT ATC GGT TGC GAA TA-3′. The ends were cut with the restriction enzymes HindIII and XbaI and ligated into pBluescript II KS-.
Xbai and hindiii restriction enzymes
Manufactured by New England Biolabs
XbaI and HindIII are type II restriction enzymes that recognize and cleave specific DNA sequences. XbaI recognizes and cleaves the palindromic DNA sequence 5'-TCTAGA-3', while HindIII recognizes and cleaves the palindromic DNA sequence 5'-AAGCTT-3'. These enzymes are commonly used in molecular biology applications, such as DNA cloning, fragment analysis, and genetic engineering.
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Lab products found in correlation
3 protocols using xbai and hindiii restriction enzymes
1
Cloning the aaap Locus from Tick-Borne Pathogens
2
Cloning of Anaplasma Locus from Infected Cells
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DNA isolated from the St. Maries and Virginia strains was digested with XbaI and HindIII restriction enzymes (New England Biolabs) and separated on a 0.7% agarose gel. Gel slices corresponding to the appropriate size of the aaap locus were extracted using the QIAquick Gel Extraction kit (Qiagen). The DNA was ligated with XbaI-HindIII-digested pBluescript II KS- (Stratagene) vector. Transformed colonies, grown in E. coli TOP10 or Stbl2 (Invitrogen) cells, were screened for inserts containing the aaap locus by membrane hybridization using the Digoxigenin-labeled aaap probe. Membrane hybridization was carried out as directed (Roche Applied Science) with denaturation in 0.5 M NaOH, 1.5 M NaCl for 15 min, neutralization in 0.5 M Tris [pH 7.5], 1.5 M NaCl for 15 min, followed by 10 min in 2× SSC. Hybridization was as described for Southern blots. Cloning of the St. Maries aaap locus from DNA isolated from infected ISE6 cells was performed via Polymerase Chain Reaction (PCR) using primers 5′-CAG GCC CAA AAT CGC GTC ATC C-3′ and 5′-CCC TAG CCC TAT ATC GGT TGC GAA TA-3′. The ends were cut with the restriction enzymes HindIII and XbaI and ligated into pBluescript II KS-.
3
In-frame deletion of RSP_1566 gene
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An in-frame, markerless deletion of RSP_1566 was constructed using the suicide vector pK18mobsacB (69 (link)). Briefly, RSP_1566 plus ~1-kbp flanking DNA up- and downstream of RSP_1566 was amplified from R. sphaeroides genomic DNA with primers g1566_F_XbaI and g1566_R_HindIII. The amplified products were digested with XbaI and HindIII restriction enzymes (New England Biolabs) and ligated into pK18mobsacB to generate pK18mobsacB_genomicRSP1566. The RSP_1566 coding sequence was deleted from pK18mobsacB_genomicRSP1566 by PCR with phosphorylated primers RSP_1566_deletionF and RSP_1566_deletionR. See Table S7 for primer sequences. The resulting PCR product was ligated to generate pK18mobsacB_ΔRSP1566 and transformed into E. coli DH5α. pK18mobsacB_ΔRSP1566 was mobilized into R. sphaeroides via conjugation with E. coli S17-1. Transconjugants were selected on LB kanamycin plates under aerobic conditions. Colonies were streaked for purity, resuspended in LB, and plated on LB sucrose (10% [wt/vol]) plates. Isolated colonies were patched onto LB kanamycin and LB sucrose plates to screen for cells that had lost the plasmid. Kanamycin-sensitive, sucrose-resistant colonies containing the desired gene deletion were identified by PCR. Gene deletion was confirmed by Sanger sequencing of the genomic region.
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