Cells were cultured at 37 °C (5% CO2) in DME-HG supplemented with 10% FCS, L-glutamine (2 mM), penicillin (100 U ml−1) and streptomycin (100 μg ml−1). One day after seeding (1 × 106 cells per well; 6-well plate), cells were transfected with 4 μg of plasmid DNA using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. NIH3T3 cells (ATCC; #CRL-1658) were collected either 48 h or 5 days after transfection to detect the expression of the gRNAs or the generation of genomic editing, respectively. For cell infection, 293T cells (ATCC; # CRL-3216) were transfected with lentiviral constructs together with ecotropic packaging plasmids using the protocol described above. Media containing viruses were collected 48 h after transfection and used to infect NIH3T3 cells. Infected cells were selected with puromycin (2 μg ml−1) for 3 days and then collected for further analysis. Analysis of proviral integrity was done by PCR using primers F2 and R2 (Supplementary Fig. 3b ). In cells displaying detectable levels of proviral recombination the amplicon corresponding to the recombined viral genome was cloned into Topo Blunt II (Invitrogene) and six clones of the resulting bacterial clones sequenced.
Topo blunt 2
Manufactured by Thermo Fisher Scientific
Topo Blunt II is a cloning vector designed for the blunt-end ligation of PCR products. It provides a simple and efficient method for cloning DNA fragments with blunt ends.
Automatically generated - may contain errors
3 protocols using topo blunt 2
1
Transfection and Lentiviral Infection of NIH3T3 Cells
2
Lentiviral Infection and Genomic Editing Protocol
3
Recombinant IspF Protein Production
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The gene encoding BcIspF was amplified from B. cenocepacia genomic DNA (strain J2315) by PCR using 5′- CAT ATG GAC TTC AGA ATC GGA CAA GG −3′ and 5′- GGA CCT CAG CCG CCC TGC TTC ACC −3′ as the forward and reverse primers respectively (Thermo Scientific). The PCR product was ligated into TOPO-Blunt-II (Invitrogen) then subcloned into a modified pET15b vector (Novagen), which produces a histidine-tagged protein with a Tobacco Etch Virus (TEV) protease site. A single nucleotide error in the forward primer gave a single amino acid mutation F3V. The gene encoding IspF from P. falciparum strain 3D7, [GenBank:XP_001349603], was synthesized (Genscript) with codons optimized for recombinant expression in E. coli. The sequence encoding residues 1–59, a predicted apicoplast targeting sequence, was excluded and the codon for Cys60 was replaced with one for serine. This synthetic gene was sub-cloned into the same modified pET15b vector as Bc ispF. Experimental procedures for recombinant protein production using E. coli BL-21 (DE3) Gold following published methods [19 (link)].
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