Journal: Nucleic Acids Research

Article Title: Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51

doi: 10.1093/nar/gkl843

Figure Lengend Snippet: PCR ( A ), Southern blot ( B ) and sequence analysis ( C ) of the selected zeocin-resistant clones. (A). Aliquots from three different clones obtained from cells that expressed wt IN (wt 1–3 ), D116A IN (D116A 1–3 ), or no IN (-IN) were subjected to total genomic DNA extraction and PCR using 5′-U3-Zeo and 3′-U5-Zeo primers (B). Total genomic DNA was digested by BamH1 restriction enzyme and submitted to Southern blot analysis with (5′- 32 P) 5′-U3-Zeo as a probe. M: DNA size marker (bp). (C) Total genomic DNA was cut by BamH1 restriction enzyme, ligated, amplified using 5′-U3-junction and 3′-U5-junction primers and sequenced using the same primers. The name of the disrupted ORF is mentioned as well as the integration site inside the gene. The 5 bp repeats are underlined, dotted lines indicate base deletions at the integration site. ( D ) The sequences of the integrated products obtained either in an in vitro concerted integration assay [data obtained from Ref. ( 17 )] or from the in vivo yeast integration system were compared. The number of duplications and deletions in both systems are reported. 'Other duplications' indicate the number of cases that exhibited duplications comprising sizes from 3 to 6 bp.

Article Snippet: Southern blot analysis was performed on genomic DNA according to a previously described protocol ( ) and using BamH1 restriction enzyme (PROMEGA) and the 32 P-labeled ODN, 5′-32 P-U3-Zeo described above as probe.

Techniques: Polymerase Chain Reaction, Southern Blot, Sequencing, Clone Assay, DNA Extraction, Marker, Amplification, In Vitro, In Vivo

Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

Article Title: Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

doi: 10.1002/0471142727.mb1216s103

Figure Lengend Snippet: Restriction map of a FLASH plasmid harboring an extension unit Extension units are released from their plasmid vectors using a quadruple digest. In the example shown, the extension unit contains coding sequence for four TALE repeats (colored rectangles). Digestion of the plasmid with BbsI and BamHI releases the unit from the plasmid with appropriate overhangs for use in the FLASH assembly method. Digestion of the plasmid with the additional restriction enzymes XbaI and SalI prevents the released unit fragment religating back into the vector, thereby eliminating the need to gel purify the fragment.

Article Snippet: ) 100X Bovine Serum Albumin (BSA) (10 mg/ml) (NEB cat. no. B9001S) Restriction enzymes (NEB): BamHI-HF (cat. no. R3136L) XbaI (cat. no. R0145L) BbsI (cat. no. R0539L) SalI-HF (cat. no. R3138L).

Techniques: Plasmid Preparation, Sequencing