Pgem t kit

Genomic DNA was isolated either from the indicated cell lines or from formalin-fixed paraffin-embedded nevi, primary or metastatic melanoma patient samples. For melanoma cell lines, cell pellets were incubated at 50°C overnight in 100 mM NaCl, 10 mM Tris–HCl, pH8, 25 mM EDTA, 0.5 SDS and 0.1 mg/ml Proteinase K and the genomic DNA was extracted with phenol-chloroform. For paraffin-fixed tissues, tissue sections scraped from five 10-μm samples were incubated at 60°C overnight (in SSC buffer, 180 mM NaCl, 0.45% SDS, 2 mg/ml Proteinase K and 1 mM DTT) and the DNA was extracted with phenol–chloroform. A 227 bp fragment including pre-miR146a was amplified by PCR using the primers listed in Supplementary file 1E 5, cloned using pGEM-T kit (Promega), and plasmid DNA isolated from 24 bacterial colonies were sequenced. Similarly, fragments of BRAF and NRAS were amplified for genotyping using primers listed in Supplementary file 1E from genomic DNA isolated from patient-derived samples, cloned using pGEM-T kit (Promega), and plasmid DNA isolated from 24 bacterial colonies were sequenced using S6 primers. All the sequencing was performed using Sanger sequencing method that typically have the error rate that range from 0.001 to 1% (Hoff, 2009 (link)).

For CmMAX1 degenerate primers were developed based on MAX1 amino acid sequences from Arabidopsis, tomato, poplar, soy, Medicago, vine and RicinusS1 Table. Furthermore, a BLAST search was done to the chrysanthemum transcriptome database of Xu et al. [62 (link)] using Arabidopsis genes known to be involved in branching (corresponding accession numbers are indicated in Table S.2). This resulted in orthologous sequences for CmMAX3, CmDRM1, CmSTM, CmRR1, CmHK3a, CmHK3b, CmAXR1, CmAXR2, CmIAA16, CmAXR6, CmIAA12, CmPIN1, CmTIR1 and CmTIR3. Reference genes were identified by BLAST against the NCBI database. These included CmACT2, CmATUB, CmUBQ10, CmUBC, CmEF1α, CmCACS, CmEXP5, CmEXP6, CmPGK, CmPSAA and CmHH3. Sequences of CmBTUB_AB608732 and CmMTP_AB542716.1. were available for Chrysanthemum. Primers S2 Table. were developed using Primer3 PLUS. Genes were cloned using a pGEM-T kit (Promega) and sequenced according to the protocol of the Big Dye Terminator Cycle Sequencing kit version 1.1 on an ABI Prism 3130 xl Genetic analyser (Applied Biosystems). BlastX [63 (link)] was used to validate isolated fragment identity.

As previously described, a qPCR assay targeting the ompB gene (9 (link)) was used. Serial dilutions of plasmids (pGEM-T kit; Promega, United Kingdom) used as external controls served for quantification (duplicates), and plasmid copy numbers were calculated using the Quant-iT PicoGreen kit (Invitrogen, USA) according to the manufacturer's instructions.
Bacterial loads were estimated with the following formula: number of R. typhi DNA copies per ml of blood = [(number of copies per PCR mixture using 1 μl DNA template)/2] × 100. Numbers of copies per reaction were calculated using serial plasmid dilutions as external standards (10³ to 10⁰ copies/μl), resulting in numbers of copies per μl DNA-eluate (Rotor-Gene 6000 software; Qiagen). The factor 2 adjusts for the 2:1 ratio of blood to DNA-eluate (resulting in numbers of copies per μl buffy coat); the factor 100 corrects for the buffy coat fraction, which makes up ∼10% of the total blood sample (5 ml total collected blood sample, ∼500 μl collected buffy coat fraction [29 (link)]).