Article Title: Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation
Figure Lengend Snippet: Generation of recq2 null mutants in bloodstream form T. brucei . ( A ) PCR was used to amplify a 5’ and a 3’ region (blue arrows) of the RECQ2 ORF (orange arrow). These PCR products were the ‘targeting regions’ cloned into the constructs shown below,which weremade to generate heterozygous (+/-) and knockout (-/-) mutants by replacing the ORF with resistance cassettes. Generalised representation of the linearised blasticidin and neomycin (G418) resistance constructs relative to the RECQ2 ORF (orange) and the targeting flanking regions (UTR; grey), which allow HR-mediated exchange (crosses). β/α tub, β/ α tubulin intergenic region; actin IR, actin intergenic region; BSD , blasticidin resistance; NEO , neomycin (G418) resistance. Not to scale. ( B ) The upper diagram shows diagnostic PCRs to confirm replacement of RECQ2 with the knockout constructs (positions of primers are indicted by arrows): a region of the ORF was amplified (“ORF PCR”) and, in addition, a region was amplified using a forward primer lying upstream of the 5’ UTR region in the knockout construct and a reverse primer specific to the BSD or NEO genes (“BSD PCR” and “NEO PCR”, respectively). The lower diagram shows agarose gels of PCR products generated from genomic DNA from wild type (WT) cells and heterozygote (+/-) and knockout (-/-) clones using primers described above. Distilled water was used as a negative control (-). Broken lines indicate different images aligned in this figure; size markers are shown (bp). ( C ). RT-PCR confirmation of recq2 mutants. A region of the PIF6 ORF and a region of the RECQ2 ORF was PCR-amplified using cDNA (+RT) synthesised from RNA extracted from wild type (wt) cells and from recq2 +/- and recq2 -/- mutants; control samples in which no reverse transcriptase had been added (-RT) are shown, as are controls using distilled water rather than substrate (-); size markers on the agarose gels are shown (bp). DOI: http://dx.doi.org/10.7554/eLife.12765.004
Article Snippet: RNA was extracted from the cells using the Qiagen RNeasy kit, and cDNA synthesis was performed using random primers and the Primer Design Precision nanoScript Reverse Transcription kit (Primer Design), according to manufacturer’s instructions.
Techniques: Polymerase Chain Reaction, Clone Assay, Construct, Knock-Out, Diagnostic Assay, Amplification, Generated, Negative Control, Reverse Transcription Polymerase Chain Reaction