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1.
Figure 6

Figure 6. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

A restriction map of pDDB78 plasmid generated using the plasmapper program (http://wishart.biology.ualberta.ca/PlasMapper/) indicating the location of unique restriction sites.

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.
2.
Figure 1

Figure 1. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

PCR amplification of the mini blaster cassette from the pDDB57 plasmid. Grey boxes in the diagram indicate the 200 bp repeats flanking the URA3 ORF. In the gel picture, lanes 1 to 5 show the ~2000 bp long cassette and a ~750 bp long loop-out product.

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.
3.
Figure 2

Figure 2. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

Schematic showing the mini blaster cassette from the plasmid pDDB57. Outcomes of subsequent transformation events are indicated step-wise in the figure. Dotted lines indicate the 100 bp long regions in the primer sequences bearing homology to the gene locus of interest (YFG1 = Your Favourite Gene 1)

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.
4.
Figure 3

Figure 3. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

Colony PCR based detection strategy for heterozygous mutants indicating the positions of detect primers (FD1 and RD1). Shown in the gel picture are colony PCRs from 14 independent transformants (lane 1 to 14). Plus indicates positive control band in the reference strains encompassing an ORF of size ~1500 bp. Presence of the wild type ORF band (~1500 bp) and a gene disruption band (~750bp) indicates a heterozygous mutant; as observed in lanes 7, 9 and 12.

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.
5.
Figure 4

Figure 4. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

Colony PCR based detection for putative homozygous mutants indicating the positions of detect primers (FD2 and RD1). Lanes 1 to 7 show PCR analysis on independent colonies. Two possible situations indicated by desired and alternative outcome are shown in the diagram. In the gel picture, lanes marked 4 and 7 indicate an expected outcome situation where the previously undisrupted allele is replaced by the cassette leading to loss of a wild type detect band (refer to the positive control lane).

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.
6.
Figure 5

Figure 5. From: Mini-blaster-mediated targeted gene disruption and marker complementation in C. albicans.

Genomic DNA confirmation for putative homozygous mutants indicating the positions of detect primers (FD1, FD2 and RD1) in the diagram. Lanes marked 1 to 8 represent transformants that yielded a negative result in the previous step. Detection strategy I using primers FD1 and RD2 confirms loss of the ~1500 bp wild type ORF band and presence of a gene disruption band (~750 bp). Note that we observe two closely spaced gene disruption bands as a second primer set, F2-R2, was used to disrupt the second allele. Size differences between the double bands should be 200 bp. Detection strategy II using primers FD2 and RD1 additionally confirms loss of the wild type band (~500 bp). Plus sign indicates corresponding positive controls using reference strain genomic DNA. Minus sign indicates no template controls.

Shantanu Ganguly, et al. Methods Mol Biol. ;845:19-39.

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