Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: In PCR #1, a 500 bp test sequence and 225 bp SV40PA sequence are added to the same reaction to form a test sequence-SV40PA combined construct. In PCR #2, the PCR #1 product and a 600 bp CMV Promoter are added to the same reaction to form the full minigene construct. Overlapping, homologous regions of these gene fragments allow for assembly to occur.

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Sequencing, Construct

Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: gBlocks and other primers used in laboratory protocols

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Sequencing, Multiplexing, Amplification

Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: PCR #1 set up. Repeat for as many gBlocks as are required.

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Concentration Assay, Sequencing, Construct

Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: 15 lanes containing independent PCR #1 products are shown. Each band is approximately 650-700 bp in size, and represents an individual test sequence (500 bp) attached to a SV40PA signal (225 bp). Note that products seem to be migrating to the same positions in pairs, as would be expected with reference and alternate containing test sequences that have identical lengths.

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Sequencing

Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: PCR #2 set up. Repeat for as many gBlocks as are required.

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Concentration Assay

Journal: Current protocols in human genetics

Article Title: In vivo and In vitro methods to identify DNA sequence variants that alter RNA Splicing

doi: 10.1002/cphg.60

Figure Lengend Snippet: Amplification of CMVp-N500

Article Snippet: Tris EDTA Suspension Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) Nuclease-Free H20 (ThermoFisher Scientific Cat. No. AM9932) Primers for PCR #1 reaction: N500-RTPCR-F2, SV40PA-N500R Phusion High-Fidelity PCR Master Mix with GC Buffer (NEB Biolabs Cat. No. M0532S) Agencourt AMPure XP Beads (Beckman Coulter {"type":"entrez-nucleotide","attrs":{"text":"A63882","term_id":"3717428","term_text":"A63882"}} A63882 ) 80% Ethanol in Nuclease-Free H20 10mM Tris Acetate, pH 8.0 - made from Trizma base (Sigma T1503) and acetic acid (Sigma 537020) D1000 ScreenTape (Agilent 5067-5582) and Reagents (Agilent 5067-5583)

Techniques: Amplification, Concentration Assay, Sequencing