To optimize for visualization of CR phenotypes, solid medium was prepared using 1.5 ml of 2% stock solution of CR per 100 ml ATGN. Plates also contained 300 μg/ml Km for selection. 100 μl of cell suspensions were plated on ATGN-CR-Kan plates at 1:100 dilution for CDGS− Upp+ ΔpruA ΔuppY, and 1:1000 dilution for CDGS− Upp+ ΔpruA ΔuppW. Plates were incubated 48-72 h and screened for decreased CR (DCR) colony phenotypes. DCR colonies from the different strain backgrounds were picked, streaked or patched on fresh ATGN-CR-Kan plates and incubated at 28°C. PCR was used to confirm that the DCR mutants were A. tumefaciens and not KmR contaminants with a DCR phenotype. Biofilm assays on randomly selected DCR mutants showed deficiencies to varying degrees. Confirmed DCR strains were inoculated in 2 ml ATGN-Kan, and 250 μl from each stationary-phase culture was pooled together into 1 ml batches. This was done separately for the two A. tumefaciens strain backgrounds. gDNA was extracted from pooled DCR cultures from the two strain backgrounds using (Promega gDNA extraction kit) and stored at 4°C. Over 50,000 colonies were screened collectively in both strain backgrounds, and roughly 0.5% were DCR mutants in each background.
Gdna extraction kit
Manufactured by Promega
The GDNA extraction kit is a laboratory tool designed to isolate genomic DNA (gDNA) from a variety of sample types. It utilizes a simple and efficient protocol to extract high-quality gDNA suitable for downstream applications such as PCR, sequencing, and genotyping.
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Lab products found in correlation
2 protocols using gdna extraction kit
1
Screening for decreased Congo red phenotypes
2
Kangaroo Endogenous Retroviral Phylogenetics
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DNA extractions (Supplementary Table S2 ) were performed using standard phenol chloroform extraction30 or the gDNA extraction kit (Promega). Conditions for PCR screening are given in the Supplementary Information. The amplicon size difference between species for each primer pair (visualized by gel-electrophoresis) was used to predict informative phylogenetic markers. The selected markers were verified by Sanger sequencing to validate the presence of Kangaroo Endogenous RetroViral Element-1 (KERV-1) insertions and target site duplications27 (link). Representatives from multiple taxa following insertion and one or more taxa without the insertion were Sanger sequenced to verify PCR patterns for all markers. For closely related species within lineages, Sanger sequences and gel electrophoresis presence of a ‘filled site’, exemplified by a ~400 nt larger PCR product, was used to establish the phylogenetic position of the marker (Supplementary Material). When direct sequencing was problematic, PCR products were ligated into the pDrive plasmid (Qiagen) prior to sequencing. All sequences were visually inspected and aligned in Se-AL 2.031 .
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