Samples of the proximal, middle, and distal colon from the same animal were collected separately. A 0.5–1.0-cm section of each intestinal region was dissected open, washed gently with distilled water to remove gut contents, and the mucus was scraped off and collected. DNA was extracted from these samples using the phenol-chloroform method. Bacterial 16S rRNA genes were amplified using the universal primers 27f (5’-AGAGTTTGATCCTGGCTCAG) and 1522r (5’-AAGGAGGTGATCCA(A/G)CCGCA) [19 (link)]. PCR products were ligated into pGEM-T vectors (Promega, Madison, Wisconsin, USA) and transformed into JM109 competent Escherichia coli (Promega). After the standard blue–white screening, clones were checked for the correctly sized insertion and submitted to the National Yang-Ming University VYM Genome Research Center (Taipei, Taiwan) for sequencing.
Jm109 competent escherichia coli
Manufactured by Promega
Sourced in United States
The JM109 competent Escherichia coli is a strain of laboratory-prepared bacterial cells that can be used for the transformation of DNA. It is a commonly used host strain for various cloning and expression applications.
Automatically generated - may contain errors
2 protocols using jm109 competent escherichia coli
1
Gut Microbiome Profiling via 16S rRNA
2
Cloning and Sequencing of Notch Alleles
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PCR products were purified by gel electrophoresis (1% agarose gel), excision of the band, and gel purification (QIAquick Gel Extraction Kit, Qiagen). Purified PCR products were ligated into the pGEM-T Easy plasmid (Promega) and transformed into JM109 competent Escherichia coli (Promega) according to standard protocols. Blue/white screening was used to identify colonies with plasmids carrying inserts. Cells from individual white colonies were grown in Luria Broth with ampicillin overnight. Plasmids were isolated from cultures using the QIAPrep Spin Miniprep Kit (Qiagen) and sequenced (ABI BigDye3.1 and ABI 3730, Applied Biosystems) with T7, SP6, M13, and/or Notch -specific PCR primers. The RAL lines and sc inversion lines were genotyped by sequencing 7–10 independent clones with most lines having at least 10 clones and some lines having hundreds of clones sequenced as part of quality control (QC) experiments. The classical Notch alleles, X balancers, the wopa23, RAL-100, and RAL-105 lines were genotyped by sequencing three to seven clones, and many were also confirmed by sequencing a PCR-amplified genomic DNA.
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