The biotin incorporation assay was performed as described (Radecke et al. 2006b (link)) with minor modifications. The EGFP reporter cells were transfected with internal biotin-labeled ODNs (BFP_S90_Biotin) and flow-sorted as described above. The genomic DNA from 1 × 105 BFP-positive cells was prepared using a Puregene Cell kit (Gentra) with a 60-min centrifugation step after isopropanol precipitation. The genomic DNA was digested to completion with XhoI and XbaI restriction enzymes (New England Biolabs). The biotinylated DNA fragments were isolated using a Dynabeads Kilobase BINDER kit (Life Technologies) according to the manufacturer's protocol, except that all reagents were supplemented with 0.1% BSA, and two additional washes using 0.1 M NaOH and 0.05 M NaCl at room temperature and one more rinse with 95°C water were performed to remove the noncovalently linked genomic fragments. The biotinylated genomic fragments were detected with a 40-cycle PCR using Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Scientific). The genomic DNA preparation prior to Dynabeads purification was used as control in a 25-cycle reaction.
Xhoi and xbai restriction enzymes
Manufactured by New England Biolabs
Sourced in United States
XhoI and XbaI are type II restriction enzymes that recognize and cleave specific DNA sequences. XhoI recognizes and cleaves the palindromic DNA sequence 5'-C^TCGAG-3', while XbaI recognizes and cleaves the palindromic DNA sequence 5'-T^CTAGA-3'. These enzymes are commonly used in molecular biology techniques such as DNA digestion, cloning, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads kilobasebinder kit, by Thermo Fisher Scientific (1 mentions)
Phusion hot start 2 high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Pmirglo dual luciferase plasmid, by Promega (1 mentions)
Jm109 cells, by Promega (1 mentions)
Applied biosystems 3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
3 protocols using xhoi and xbai restriction enzymes
1
Biotinylated DNA Enrichment and Detection
2
PD1 3' UTR Cloning and Luciferase Assay
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The 3’ UTR of PD1 was amplified from wild-type c57BL/6 lymphocyte cDNA using PCR with 10 μM PD1 3’ UTR forward and reverse primer:
Forward: 5’-ATATACTCGAGCCAGATTCTTCAGCCATTAGCATGCT
Reverse: 5’-GCGTGTCTAGATTTAAAGCTTTTGGTACCATTTAATTATAACGGGCT and Taq polymerase (Invitrogen). The amplified cDNA was separated on a 1.5% agarose gel and the QIAquick Gel Extraction Kit (Qiagen) was used to isolate the PD1 3’ UTR cDNA. The PD1 3’ UTR and pmirGLO Dual Luciferase Plasmid (Promega, USA) were cleaved with XhoI and XbaI restriction enzymes (New England Biolabs, Ipswich, MA) and allowed to ligate overnight at 16° C. The PD1 3’ UTR pmirGLO Dual Luciferase Plasmid was amplified in JM109 cells (Promega, Madison, WI) and extracted using the GeneJET Plasmid Miniprep Kit (Fermentas, Burlington, ON). The concentration of the plasmid was then measured using a NanoDrop ND-1000. Sequencing for confirmation of successfully ligation was done using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) in an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems).
Forward: 5’-ATATACTCGAGCCAGATTCTTCAGCCATTAGCATGCT
Reverse: 5’-GCGTGTCTAGATTTAAAGCTTTTGGTACCATTTAATTATAACGGGCT and Taq polymerase (Invitrogen). The amplified cDNA was separated on a 1.5% agarose gel and the QIAquick Gel Extraction Kit (Qiagen) was used to isolate the PD1 3’ UTR cDNA. The PD1 3’ UTR and pmirGLO Dual Luciferase Plasmid (Promega, USA) were cleaved with XhoI and XbaI restriction enzymes (New England Biolabs, Ipswich, MA) and allowed to ligate overnight at 16° C. The PD1 3’ UTR pmirGLO Dual Luciferase Plasmid was amplified in JM109 cells (Promega, Madison, WI) and extracted using the GeneJET Plasmid Miniprep Kit (Fermentas, Burlington, ON). The concentration of the plasmid was then measured using a NanoDrop ND-1000. Sequencing for confirmation of successfully ligation was done using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) in an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems).
3
Yeast Complementation Using AHA2 Variants
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The constructs used to express WT AHA2, ahaT947A, aha2∆30, aha2∆44, and aha2∆57 for yeast complementation were generated based on the plasmid YEp351. The constitutive yeast native PM H+-ATPase promoter PMA1 was employed to regulate the expression of the genes. The target gene fragments were amplified by PCR using phusion high-fidelity DNA-polymerase (NEB). Both the PCR products and YEp351 were digested with XhoI and XbaI restriction enzymes (NEB) and then ligated by the T4 DNA ligase (NEB). The inserted genes were fully sequenced.
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