Journal: Scientific Reports

Article Title: Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells

doi: 10.1038/srep36341

Figure Lengend Snippet: TEM characterization of the Sep/DNA bionanocomposite ( A–C ). ( D,E ) TEM images of a single molecule of Sep/DNA. The DNA bound to the sepiolite at the edge of the nanofiber and on its external surface. Nanofibers can be completely coated with DNA, but some DNA molecules are bound at one of their extremities, and two different nanofibers can be linked by a DNA plasmid. Reaction conditions: 1 mg/ml sepiolite, 10 mM Tris-HCl, pH = 7.5, 5 mM MgCl 2 , and 50 ng·μl −1 plasmid DNA (5.7 kb). ( G ) Scheme illustrating the DNA-sepiolite bionanocomposite resulting from the sepiolite interaction with linear DNA. It is also show the silanol groups (Si-OH) present at the external surface of the sepiolite fibers which are in hydrogen bonding interaction with nitrogenated bases at the DNA chains.

Article Snippet: Different DNA molecules were used: a) a commercial preparation of low molecular weight DNA, from salmon sperm, was supplied by Sigma-Aldrich (31149–50G-F, lot# 0001393538); the lyophilized DNA was dissolved in 10 mM Tris-HCl, pH = 7.5, to produce a 1.9 mg·ml −1 solution. b) A circular DNA plasmid (5.7 kb, pCMV) was obtained by the amplification of a bacterial culture and was purified using the commercial kit from Macherey-Nagel; DNA PUC19 plasmid was supplied by New England BioLabs at 1 mg ml −1 (pUC19 Vector #N3041S, lot# 0361204. c) Single-stranded DNA custom oligos 15 nt long (ss-Oligo) were provided by Eurogentec (M13 sense 5′-gtaaaacgacggcca-3′ and M13 antisense 5′-tggccgtcgttttac-3′); the lyophilized solids were dissolved in MilliQ water to provide respective solutions of each single-stranded DNA sense. d) Double-stranded DNA oligos 15 bp long (ds-Oligo) were produced using an annealing reaction in the presence of 10X NE Buffer provided by New England Biolabs and heating for 3 min at 95 °C, following by a slow decrease in temperature, after which the buffer was changed to Tris-HCl 10 mM pH = 7.5 using the Micro Bio-Spin TM chromatography columns.

Techniques: Plasmid Preparation

Journal: Malaria Journal

Article Title: Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

doi: 10.1186/1475-2875-10-119

Figure Lengend Snippet: Replacement strategy of mutant Pfdhfr-ts into Pbdhfr-ts by double cross-over homologous recombination . (A) endogenous Pbdhfr-ts gene locus in PbGFP wild-type parasite, (B) linearized plasmid pY005 containing mutant Pfdhfr S108N , (C) Pfdhfr S108N -ts gene locus in transgenic PbPf S108N parasite. Positions of primers used for PCR amplification are indicated by arrows. The expected size of PCR products are described.

Article Snippet: The merozoites were transfected with the circular plasmid DNA harboring Pfdhfr mutant libraries using the standard Amaxa Nucleofector protocol [ ] and re-infected into animals by i.v. injection.

Techniques: Mutagenesis, Homologous Recombination, Plasmid Preparation, Transgenic Assay, Amplification

Journal: Malaria Journal

Article Title: Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

doi: 10.1186/1475-2875-10-119

Figure Lengend Snippet: PCR and RT-PCR analysis of the transgenic mutant parasite . (A) PCR analysis of 5' and 3' integrations on genomic DNA isolated from transgenic mutant parasites, PbPf S108N, are shown in lanes 1 and 4, respectively. Genomic DNA isolated from PbGFP wild-type parasite and distilled water (Neg) served as negative controls as shown in lanes 2, 5 and 3, 6, respectively. (B) RT-PCR analysis of PbPf S108N parasites. RNA from the PbPf S108N parasite was reverse transcribed to cDNA and used as a template to amplify Pfdhfr, Pbdhfr and P. berghei alpha tubulin ( Pbα-tubulin ) genes. The reactions were performed with reverse transcription (lane 1), without reverse transcription (lane 2), P. berghei cDNA derived from PbGFP and distilled water (Neg) were used as negative controls with and without reverse transcription (lanes 3-6).

Article Snippet: The merozoites were transfected with the circular plasmid DNA harboring Pfdhfr mutant libraries using the standard Amaxa Nucleofector protocol [ ] and re-infected into animals by i.v. injection.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Mutagenesis, Isolation, Reverse Transcription, Derivative Assay