T4 dna polymerase

For the synthesis of aminoallyl RNA (aaRNA) probes, total RNA was extracted from each biological replicate (seedling) separately. Total RNA was precipitated (4M LiCl), residual genomic DNA was hydrolyzed with DNaseI (Fermentas, St. Leon-Rot, Germany) and purified with the NucleoSpin RNA Clean-up Kit (Macherey-Nagel, Düren, Germany). For first strand cDNA synthesis, 5 μg of total RNA and the Superscript II from Invitrogen (Life Technologies, Carlsbad, USA) were used. Second strand synthesis was performed using DNA Polymerase I and RNase H followed by a 5 min incubation step of T4 DNA Polymerase (Fermentas, St. Leon-Rot). Residual RNA was hydrolyzed with RNase A. In-vitro transcription was performed with the T7 RNA Polymerase (Fermentas, St. Leon-Rot, Germany) for 4h to incorporate aminoallyl-labeled UTPs (Fermentas, St. Leon-Rot, Germany). Finally, residual DNA was degraded with DNase I (Fermentas, St. Leon-Rot, Germany).
Synthesized aaRNA was coupled with fluorescence dyes Cy3 or Cy5 (GE Healthcare, Chalfont St. Giles, UK). The RNeasy MinElute Kit (Qiagen, Hilden, Germany) was used for purification and removal of unbound dye.

After reverse transcribing tRNA to cDNA, second-strand synthesis was performed using RNase H, DNA Polymerase I, and T4 DNA Polymerase (Fermentas) according to the manufacturer’s recommended procedure. The double-stranded cDNA was then purified using a HiPure PCR Kit (Magen). A sequencing library was constructed using the NEBNext Ultra DNA Library Prep Master Mix Set for Illumina (NEB). Next-generation sequencing was performed on an Illumina HiSeq-2000 sequencer for 100 cycles. High-quality reads passing the Illumina filter were kept for subsequent data analysis. The sequencing dataset was deposited in the Gene Expression Omnibus database under Accession Number GSE62995.
Adapter sequences were trimmed from the reads. The reads were aligned to tRNA reference sequences using the modified Smith–Waterman algorithm that considered modified nucleotides matching any nucleotide [43 ]. A maximum of 3 errors (including mismatches, insertions, and deletions) was allowed in the alignments, and the minimal alignment length was set to 25 bp. The number of aligned tRNA reads were then normalized using the spike-in reads.

FideliTaq PCR Master Mix (USB, USA) was used for DNA amplification. T4 DNA ligase, T4
DNA polymerase, Klenow fragment, T4 polynucleotide kinase, shrimp alkaline
phosphatase (SAP), restriction enzymes and the protein molecular mass marker were
purchased from Fermentas (USA). The HiTrap Chelating HP column was purchased from GE
Healthcare (Uppsala, Sweden). Chitin from crab shells and Fluorimetric Chitinase
Assay Kit were purchased from Sigma-Aldrich (USA). Polyglucosamine (DA 0%) used for
the preparation of partially acetylated chitosans as well as α- and β-chitin were
kindly provided by Mahtani Chitosan (India). All other chemicals used for chitinase
analysis were of analytical grade.

For an overview of the plasmids used and created in this study, see Table 1. For the primers, see Table 2. For the construction of pMD18Tslr1670, slr1670 was amplified from genomic DNA of Synechocystis with primers slr1670-BamHI and slr1670-XhoI and introduced into pMD18-T. For the construction of pMD18Tslr1670KmR, pMD18Tslr1670 was digested with NcoI, blunted with T4 DNA Polymerase and ligated with the Km resistance cassette (obtained from pRL446 Elhai and Wolk, 1988 (link) by digestion with PvuII). The glpK gene was amplified from the genomic DNA of Synechocystis by PCR with primers glpK-Fwd and glpK-Rev, and cloned into the pMD 18-T vector (Takara, Japan). The resulting plasmid was digested by NheI, blunted by T4 DNA Polymerase (Fermentas), and ligated to the blunted chloramphenicol resistance gene cassette, resulting in the plasmid pXT323.

T4 DNA polymerase and T4 polynucleotide kinase (T4 PNK) were purchased from Fermentas. Pfu DNA polymerase was purchased from Sangon Biotech. Phusion DNA polymerase and λ exonuclease were purchased from New England Biolabs. Competent DH5α cells were prepared by the transformation and storage solution [15 (link)] or purchased from Beijing CoWin Bioscience.

All chemicals used were of analytical grade. Restriction endonucleases, T4 DNA polymerase, Taq polymerase and T4 DNA ligase were obtained from MBI Fermentas. Chemicals 12-O-tetradecanoylphorbol 13-acetate (TPA) and MG132 were from Sigma-Aldrich (Stockholm, Sweden). TurboFectTM and Lipofectamine™ transfection reagents were purchased from Thermo Fisher Scientific (Uppsala, Sweden). Horseradish peroxidase-linked anti-immunoglobulins were from Dako A/S (Sundbyberg, Sweden). The monoclonal antibody C7-50 [34 (link)] recognizing HCV C was from Santa Cruz Biotechnology (Heidelberg, Germany).

Genomic DNA of passage 11 (∼275 generations) was digested with DdeI and then separated on a 0.8% agarose gel. DdeI-treated fragments shorter than 350 bp were gel-extracted. Purified fragments were blunt-end treated with T4 DNA polymerase (Fermentas) according to manufacturer’s instruction. These fragments were spin-purified and then ligated into dephosphorylated SmaI-treated pUC18. The ligation mixture was transformed into E. coli cells and transformants were selected on LB-ampicillin plates containing X-gal and IPTG. Transformants were passaged onto both a master plate and a Hybond N membrane (Amersham). In total, 10 202 colonies were screened for the presence of telomere sequences by colony hybridization with the telomeric probe. A total of 448 colonies giving hybridization signal were subjected to plasmid DNA (pDNA) purification and were further screened by slot blot hybridization, where the clones showing hybridization signal were selected for sequencing using the universal primer M13 (17-mer). Three colonies (ID 382, ID 406, and ID 448) showed a strong hybridization signal, indicating telomere-containing insert.