One shot top10 chemically competent e coli

pReceiver-M46 (C-Flag+IRES-eGFP) control, SEMA4A wild-type and SEMA4A V78M mutated vectors were purchased from GeneCopoeia and propagated in One Shot TOP10 Chemically Competent E. coli (Life Technologies). Plasmids were purified by JETSTAR Maxi Plasmid Purification Kit (Genomed) and checked by direct sequencing. One day before transfection, 6 × 10⁵ cells were seeded into six-well tissue culture plates to achieve 60 to 80% confluency. Plasmid and Lipofectamine LTX (Life Technologies) were diluted at a ratio of 1:10 (HCT-116) or 1:5 (293T), respectively, in 500 μl serum-free Opti-MEM medium (Life Technologies) for transfection. If not indicated otherwise, cells were usually grown for 48 h after transfection before whole-cell lysate preparation.

Several primer sets were designed to: 1) validate the mutations identified in exome sequencing; 2) detect mutations directly; 3) validate the splicing isoforms; or 4) confirm the effects of splicing mutation. Primers sequences and PCR conditions were available on request. Sanger sequencing was performed on a 3730 DNA analyzer (Life Technologies). PCR products were cloned when necessary by using TOPO TA Cloning Kit (Life Technologies) and One Shot TOP10 Chemically Competent E. coli (Life Technologies). Sequencher (ver. 4.7, Gene Codes) and Mutation Surveyor (ver. 4.0.6, SoftGenetics) were used for aligning sequencing chromatographs to reference sequences and mutation detection.

All truncations or point mutations in the PPP1R15A coding sequence were made as follows. Fifty nanograms of plasmid template DNA were mixed with 5 µl Pfu turbo DNA polymerase reaction buffer [10×], 1 µl Pfu turbo DNA polymerase (Agilent Technologies, Santa Clara, CA), 125 ng forward primer, 125 ng reverse primer, 1 µl of 25 mM dNTPs, made up to 50 µl with water. A PCR thermocycler was run using the following program parameters: 95°C for 30 s, 95°C for 30 s, 18 cycles (54°C for 1 min, 67°C for 20 min, 94°C for 1 min, 55°C for 1 min, 72°C for 10 min). Completed reactions were treated with 1 µl Dpn1 restriction enzyme, incubated at 37°C for 2 hr before using 5 µl of the reaction mix for a standard transformation into One Shot TOP10 chemically competent E. coli (Life Technologies, Paisley, UK).

PCR products were either directly cloned or first separated on a 1% low melting gel (Nusieve GTG Agarose, Cambrex, Rockland, USA) excised and cloned with Topo TA Cloning kit (Life Technologies Europe B.V. (Invitrogen), Zug, Switzerland) according to manufacturer’s instruction. After transformation into One Shot Top 10 chemically competent E. coli (Life Technologies Europe B.V. (Invitrogen), Zug, Switzerland) bacteria were grown on a LB-amp-X-Gal plate overnight at 37°C. Four to ten colonies were picked and grown overnight at 37°C in LB medium supplemented with 100 μg ampicillin/ml (Ampicillin-sodium salt, Carl Roth GMBH, Karlsruhe, Germany).
Plasmid DNA purification was performed with the QuickLyse Miniprep Kit (Qiagen AG, Hombrechtikon, Switzerland). The sequencing was done by Microsynth (Balgach, Switzerland). Sequences were edited and analyzed with the following software: DNASTAR software (version 5), clone manager, ClustalX 2.0.3. Phylogenetic trees were constructed with Mega (version 5.1) software
[30 (link)]. Sequence data were submitted to GenBank and were assigned the accession numbers [GenBank: KF990530 to GenBank: KF990540].

In order to obtain the standards for the RT-PCR, a PCR with the designed primers was carried out on the DNA extracted from R. conorii conorii Malish 7 strain cultured in VERO cells using the conditions previously described and an annealing temperature of 60 °C. PCR products were purified, inserted into the cloning vector pCR2.1 TOPO TA (Applied Biosystems, Waltham, MA USA), and propagated in Escherichia coli (One Shot TOP10 Chemically Competent E. coli, Life Technologies, Carlsbad, CA, USA). Plasmid DNA was extracted using the Wizard purification system SV minipreps DNA purification system (Promega, Madison, WI, USA), quantified using a Nanodrop ND1000 Spectrophotometer, and sent for sequencing (Macrogen Inc., Amsterdam, The Netherlands). To obtain a series of standards covering a range from 10 to 10⁸ copies of DNA/μL, 1:10 serial dilutions were prepared; aliquots of these dilutions were stored at −20 °C and thawed only once.