Lyophilized oligonucleotides were obtained from IDT diluted in double distilled water to a final concentration of 1 μg/μL. The oligonucleotides were then phosphorylated using T4 PolyNucleotid Kinase (NEB). The double-stranded oligonucleotide formation was carried out by rising the temperature from 37°C to 95°C during 5 min, followed by a progressive temperature reduction of 5 degrees every 30 s until reaching 25°C. The PAC154-2A-puro plasmid was cut with BbsI (NEB) and purified by gel extraction (Thermo Scientific). A double-stranded oligonucleotide diluted 1–200 (5 ng of oligonucleotide) in double-distilled water was added to the purified pAC154 plasmid. The ligation was done with the Quickligase (NEB) during incubation at 25°C for 25 min.
Gel extraction
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Gel extraction is a laboratory technique used to isolate and purify DNA or RNA fragments from agarose or polyacrylamide gels. It involves excising the desired DNA or RNA band from the gel and then extracting the nucleic acid using a buffer solution and a separation column.
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Lab products found in correlation
5 protocols using gel extraction
1
Preparation of Phosphorylated Oligonucleotides for Cloning
2
Camel Lactoferricin-Lactoferrampin Chimera
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The chimeric lactoferricin and lactoferrampin consists of 36 amino acids and was generated through the fusion of two short regions of camel lactoferrin 284DLIWKLLVKAQEKFGRGKPS303 (ID: AHJ37525) and 49RVKKMRRQWQACKSS35 (ID:NP_001290496.1) linked by Lysine (GenBank accession number: MH327768). The chimeric peptide encoding sequence was codon optimized for the appropriate expression in L. lactis by Genscript® (USA). The restriction sites of SapI and SalI were added to the N and C-terminus of the chimera sequence for cloning in pAMJ1653 vector. The sequence was chemically synthesized by Generay Biotech (Shanghai, China). For vector construction, pAMJ1653 and pGH cloning vectors (a vector harboring synthetic gene) were sequentially digested by SapI (New England Biolabs, England) and SalI restriction enzymes (Thermo Fisher Scientific, USA). The digestion products were then purified and ligated by gel extraction and fast ligation kits (Thermo Fisher Scientific, USA), respectively.
3
Cas9 Screen for HaloTag-ORF68 Fusion
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Cas9 screen was performed as the CRISPRi screen above using a Cas9+ iSLK cell line infected with a copy of BAC16 containing a HaloTag-ORF68 fusion. The library was then amplified using staggered primers and a modified amplification protocol previously described[48 (link)]. qPCR was used to determine cycle number at ¼ CT. All PCR product was run over a single Minelute column (Qiagen) for each sample. Size selection was performed using a gel extraction (Thermo). Library was sequenced on Illumina NextSeq 2000 with a 150bp single-read using Illumina sequencing primers. Adaptors were removed using cutadapt[49 (link)], and reads were aligned to library using bowtie[50 (link)].
Log2 values were calculated as above, and enrichment values were averaged from two replicates. For each exon boundary, a window of 500 bp on one side was tested against a 500 bp on the other to calculate p-values using MW test. Median values were used to determine the sign of the shift. Exons with p values < 0.001 and consistent signs were considered hits.
Log2 values were calculated as above, and enrichment values were averaged from two replicates. For each exon boundary, a window of 500 bp on one side was tested against a 500 bp on the other to calculate p-values using MW test. Median values were used to determine the sign of the shift. Exons with p values < 0.001 and consistent signs were considered hits.
4
Amplification of Biosynthetic Genes
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The genD2, genS2, genN, and genD1 genes were each amplified from the genomic DNA of M. echinospora ATCC15835 by PCR using Pfu DNA polymerase with 25 cycles of denaturing at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min plus a final extension at 72°C for 10 min. The PCR products were digested with appropriate restriction enzymes, purified by gel extraction (Fermentas), and inserted into a pET28a(+) plasmid. The resulting constructs were verified by DNA sequencing.
5
Cloning and Sequencing of Actinobacterial Genes
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Genes genQ, genB1, genB2, genB3, and genB4 were amplified from the genomic DNA of M. echinospora ATCC15835 by PCR (see List of Primers Used in Supplemental Information ). PCR was carried out using Pfu DNA polymerase with 25 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 2 min plus final extension at 72°C for 10 min. The PCR products were digested with appropriate restriction enzymes, purified by gel extraction (Fermentas), and inserted into plasmid pET28a(+). The resulting constructs (Table S2 ) were verified by DNA sequencing.
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