clai  (New England Biolabs)

Journal: Nature Communications

Article Title: Designer diatom episomes delivered by bacterial conjugation

doi: 10.1038/ncomms7925

Figure Lengend Snippet: Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.

Article Snippet: First, ClaI restriction digest or mock reaction was performed on samples using 1–2 μg total DNA in a 100-μl total reaction with 1 × CutSmart buffer and ClaI (NEB) or water in digested or mock-digested samples, respectively.

Techniques: Expressing, Selection, Isolation, Agarose Gel Electrophoresis, Derivative Assay, Marker, Plasmid Preparation, Transformation Assay, Real-time Polymerase Chain Reaction, Fluorescence

Journal:

Article Title: Initiation of DNA repair mediated by a stalled RNA polymerase IIO

doi: 10.1038/sj.emboj.7600933

Figure Lengend Snippet: Sequential assembly of NER factors onto RNA pol IIO. ( A ) Scheme depicting the reaction. Cax-Pt was transcribed and then cut by Cla I to form the Cla I-cut EC-400, which was further incubated with different combinations of four repair factors (the omitted

Article Snippet: Cax-Pt was then cut by Cla I (New England Biolabs) in a reaction volume of 40 ml.

Techniques: Incubation

Journal: PLoS Pathogens

Article Title: Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host

doi: 10.1371/journal.ppat.1006656

Figure Lengend Snippet: Confirmation of xer1 disruption in selected M . agalactiae ‘switchover’ clones via PCR and Southern analysis. (A) Verification of xer1 disruption in crude DNA extracts of selected ‘switchover’ clones by PCR using primers RecEndET28 and T3ISLrev specific to the chromosomal xer1 region and the pR3 plasmid backbone, respectively. 2 kb PCR product confirms xer1 disruption [ 13 ] in selected ‘switchover’ clones picked from the right (R)—parotideal (PAR) or—mandibular (MAN) lymph nodes of sheep MS 7 or MS 10. The observed bands were comparable to the positive controls corresponding to the crude DNA extracts (PLMY and PLMU) as well to pure DNA preparation of PLMY (+DNA); as expected this band was absent in the three negative controls corresponding to water (H 2 O), PG2 crude extract (PG2) and SP4 broth (Medium). (B) Southerns were performed as described earlier [ 13 ] whereby Cla I-digested genomic DNA was hybridized with xer1 -specific probe. xer1 disruption is confirmed in the ‘switchover’ clones (lanes 3–8) by the presence of two bands of 3.7 kb and ~18.9 kb as also seen for PLMU control (lane 2), whereas the wild type PG2 (lane 1) with intact xer1 shows a 13 kb band. Disruption plasmid pR3 (lane 9) shows the expected 10 kb band. Switchover clones: lane 3, MS 10 right udder/VpmaV; lane 4, MS 6 mesenterial LN/VpmaW; lane 5, MS 6 right iliac LN/VpmaX; lane 6, MS 6 mesenterial LN/VpmaW (PLM W); lane 7, MS 9 right udder/VpmaX (PLM X) ; lane 8, MS 6 right iliac LN/VpmaX.

Article Snippet: Confirming xer1 disruption via Southern blot analyses Mycoplasma genomic DNA was isolated by QIAamp DNA Mini Kit (Qiagen) and digested with Cla I (New England Biolabs) at 37°C for at least 6–7 h before subjecting to electrophoresis on a 1% agarose gel.

Techniques: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Mass Spectrometry

Journal: BMC Biotechnology

Article Title: Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

doi: 10.1186/1472-6750-14-16

Figure Lengend Snippet: Validation of TGR of WT- ply from IDM- ply mutant. (A) Expression of WT-PLY in the 8 h, Day 1 and Day 2 IDM- ply culture by real-time PCR (B) Cla I- digested D39, IDM- ply and AG2, which was sensitive to erm and was obtained from Day 1 culture of IDM- ply mutant, were probed with a digoxigenin-labeled ply-pb in southern blot hybridization. (C) AG2 would produce PLY whose size was the same as PLY of D39 when incubating with a rabbit polyclonal PLY antibody.

Article Snippet: Kit (Qiagen, Hilden, Germany) and were then digested by Cla I (New England Biolabs, Ipswich, MA, USA) according to the manufactures’ instruction.

Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Labeling, Southern Blot, Hybridization

Journal: BMC Biotechnology

Article Title: Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

doi: 10.1186/1472-6750-14-16

Figure Lengend Snippet: Construction of the ply and lytA knockdown mutants by IDM. Partial sequences of pneumolysin ( ply -s) (A) and autolysin ( lytA -s) (B) genes were cloned into plasmids pVA891 (A) and pEVP3 (B) , containing an erythromycin (erm)- and a chloramphenicol (cat)-resistance marker, respectively. Cloned plasmids were then used to generate homologous recombinants from their WT ply or lytA genes, respectively. Two primer sets, ply-P1/pVA891-R and ply-P2/pVA891-F or lytA-P1/pEVP3-R and lytA-P2/pEVP3-F, were used to verify the insertion of pVA891 and pEVP3 in IDM- ply (A) and - lytA (B) , respectively. C, Cla I.

Article Snippet: Kit (Qiagen, Hilden, Germany) and were then digested by Cla I (New England Biolabs, Ipswich, MA, USA) according to the manufactures’ instruction.

Techniques: Clone Assay, Marker