Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Layout of rat HoxA locus in the rn6 genome assembly. The rn6 genome includes an erroneous duplication at the HoxA locus between gaps in the assembly. The SynHoxA assemblon sequence is based on bringing together the two ‘separate’ RnHoxA segments. The sequence was segmented into 28 ∼5kb PCR amplicons with terminal homology of ∼200bp to adjacent amplicons. Conservation to the mouse genome is depicted using the multiz track from the UCSC genome browser. (B) Schematic depicting the assembly workflow for the 134kb SynHoxA assemblon. BACs containing Rat HoxA were used as PCR template to generate 28 segments tiling the entire HoxA locus. These segments were co-transformed into yeast with appropriate linkers and assembly vector to build two ∼65kb half assemblons into centromeric yeast-bacteria shuttle vectors. These half assemblons are recovered to bacteria and amplified. Full 134kb assemblon was built from half assemblons after releasing them from the vector using terminal restriction enzymes ( AsiSI ) and transforming into yeast. Full assemblon was then recovered from yeast into bacteria for amplification and verification. (C) Agarose gel of the 28 PCR amplicons that tile the 134kb SynHoxA assemblon. (D) Strategy to PCR-screen yeast colonies derived from assembly experiments. Primers (red arrows) span assembly junctions and test presence/absence of amplicons in many yeast colonies. Reproduced from ref with permission from authors. (E) Agarose gel showing one yeast colony carrying the full 134kb SynHoxA assemblon verified manually for the presence of all assembly junctions, using the strategy outlined in panel D. (F) Half and Full 134kb SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using field inversion gel electrophoresis (FIGE). Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Sequencing, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Purification, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Layout of rat HoxA locus from the rn6 genome assembly depicting genes, Rn HoxA cluster segments in black and previously identified distal enhancers in purple. The Enhancers+SynHoxA assemblon sequence is made by stringing all the enhancers directly upstream of the SynHoxA assemblon sequence. Conservation to mouse genome is depicted using multiz track from the UCSC genome browser. (B) PCR amplicons tiling enhancer sequences were generated from Rat HoxA BACs and co-transformed into a yeast strain containing the 134kb SynHoxA assemblon with a gRNA vector targeting the left terminus of the 134kb assemblon. The enhancer PCR amplicons were used to repair this break, resulting in the construction of the 170kb Enhancers+SynHoxA assemblon. Assemblon was recovered into bacteria for amplification and verification. (C) Agarose gel of the 8 PCR amplicons containing enhancer sequences. (D) Agarose gel showing one yeast colony tested for the presence of novel enhancer assembly junctions and with primers spanning 134kb SynHoxA . (E) 134kb and 170kb assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Sequencing, Generated, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Purification

Journal: bioRxiv

Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

doi: 10.1101/2021.07.07.451065

Figure Lengend Snippet: (A) Schematic of assembly strategy for 130kb RAREΔ SynHoxA and 166kb Enhancers + RAREΔ SynHoxA . Nature of the RARE mutations is shown on the right. RAR binding data comes from previously published reports. (see Methods) (B) Sanger sequencing traces confirmed precise CRISPR editing of RAREs in yeast. (C) SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Bands correspond to expected fragment lengths. (D) Sequencing data of assemblon BACs purified from E. coli aligned to a custom mm10 reference genome. Positions of the enhancers and protein coding genes are shown in black.

Article Snippet: 500 ng of lambda monocut ladder (New England Biolabs N3019S) were used as a molecular weight standard.

Techniques: Binding Assay, Sequencing, CRISPR, Purification

Journal:

Article Title: Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

doi:

Figure Lengend Snippet: Restriction patterns from AseI digests of M. avium isolates resolved by PFGE. Lanes: 1 and 6, bacteriophage lambda DNA concatemers (sizes [in kilobases] are indicated on the left); 2, isolate 100A8; 3 and 4, pattern P7 (isolates 100A28 and 100A32, respectively); 5, isolate 100A25; 7 to 11, five sequential isolates from one patient, respectively (pattern P1); 12 to 15, four isolates from two patients, respectively (pattern P2).

Article Snippet: Lanes 6 and 18, bacteriophage lambda DNA- Bst EII digest; lane 12, pBR322 DNA- Msp I digest (New England Biolabs); lanes 1 to 5, five sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P1); lanes 7, 8, and 9, three sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P2) (no amplification product was detected in lane 8 in that experiment); lane 10, isolate 100A31 from another patient (PCR profile A, PFGE pattern P2); lanes 11 and 13, two sequential isolates from one patient, respectively; lane 14, isolate 100A7 from a different patient (PCR profile E, PFGE pattern P6 or a unique pattern); lanes 15, 16, and 17, isolates 100A13, 100A26, and 100A30, respectively.

Techniques: Lambda DNA Preparation

Journal:

Article Title: Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

doi:

Figure Lengend Snippet: PCR typing of clinical M. avium isolates. Lanes: 1 to 5, 7 to 11, and 13 to 17, patterns of isolates obtained from 15 unrelated patients; lanes 6 and 18, bacteriophage lambda DNA-BstEII digest molecular weight marker; and lane 12, pBR322 DNA-MspI digest (New England Biolabs).

Article Snippet: Lanes 6 and 18, bacteriophage lambda DNA- Bst EII digest; lane 12, pBR322 DNA- Msp I digest (New England Biolabs); lanes 1 to 5, five sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P1); lanes 7, 8, and 9, three sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P2) (no amplification product was detected in lane 8 in that experiment); lane 10, isolate 100A31 from another patient (PCR profile A, PFGE pattern P2); lanes 11 and 13, two sequential isolates from one patient, respectively; lane 14, isolate 100A7 from a different patient (PCR profile E, PFGE pattern P6 or a unique pattern); lanes 15, 16, and 17, isolates 100A13, 100A26, and 100A30, respectively.

Techniques: Lambda DNA Preparation, Molecular Weight, Marker

Journal:

Article Title: Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

doi:

Figure Lengend Snippet: Electrophoretic PCR patterns for M. avium isolates. Lanes 6 and 18, bacteriophage lambda DNA-BstEII digest; lane 12, pBR322 DNA-MspI digest (New England Biolabs); lanes 1 to 5, five sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P1); lanes 7, 8, and 9, three sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P2) (no amplification product was detected in lane 8 in that experiment); lane 10, isolate 100A31 from another patient (PCR profile A, PFGE pattern P2); lanes 11 and 13, two sequential isolates from one patient, respectively; lane 14, isolate 100A7 from a different patient (PCR profile E, PFGE pattern P6 or a unique pattern); lanes 15, 16, and 17, isolates 100A13, 100A26, and 100A30, respectively.

Article Snippet: Lanes 6 and 18, bacteriophage lambda DNA- Bst EII digest; lane 12, pBR322 DNA- Msp I digest (New England Biolabs); lanes 1 to 5, five sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P1); lanes 7, 8, and 9, three sequential isolates from one patient, respectively (PCR profile A, PFGE pattern P2) (no amplification product was detected in lane 8 in that experiment); lane 10, isolate 100A31 from another patient (PCR profile A, PFGE pattern P2); lanes 11 and 13, two sequential isolates from one patient, respectively; lane 14, isolate 100A7 from a different patient (PCR profile E, PFGE pattern P6 or a unique pattern); lanes 15, 16, and 17, isolates 100A13, 100A26, and 100A30, respectively.

Techniques: Lambda DNA Preparation, Amplification