Redundant tiling PCR was performed using three separate multiplex PCR runs per sample. The primer sets for each multiplex PCR group (#1, #2, and #3) were mixed at a total concentration of 10 μM, and the forward primer was mixed with a biotinylated forward primer with the same sequence at a specified concentration (Supplementary Table 1 ). For each reaction, 1.2 μL 10× diluted cDNA template, 1.44 μL primer mix, 5 μL Q5 hot start 2× master mix (NEB), and 2.36 μL nuclease-free water were mixed and amplified with the following thermocycling conditions: 98°C for 30 s, 35 cycles of 95°C for 15 s, 63°C for 5 min, and holding indefinitely at 4°C. The PCR products of the three multiplex PCR groups for each specimen were pooled and used for NGS library preparation.
Q5 hot start 2 master mix
Manufactured by New England Biolabs
Sourced in United States
The Q5 Hot Start 2× Master Mix is a high-fidelity, ready-to-use PCR reaction mixture formulated for sensitive and specific amplification of DNA templates. It contains the Q5 High-Fidelity DNA Polymerase, dNTPs, and reaction buffer optimized for efficient DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Gibson assembly 2 master mix, by New England Biolabs (2 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
6 protocols using q5 hot start 2 master mix
1
Multiplexed Tiling PCR for NGS
2
Protease-Activated RNA Polymerase Assay
3
Molecular Cloning and Sequencing Protocol
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Unless indicated otherwise, PCRs were performed with the Q5 Hot-start 2 master mix (New England Biolabs) and cloning was performed using the In-Fusion HD cloning kit (Takara Bio) or restriction/ligation-dependent cloning. Newly introduced sequences were verified by Sanger sequencing. Oligonucleotide and gBlock sequences are listed in the SI Appendix, Table S2 .
4
DNA Cutting Assay with CRISPR
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DNA cutting assays were performed following New England Biolabs (NEB) method.39 ,40 Briefly, genomic DNA was extracted from primary isolated CD34+ cells (STEMCELL Technologies, Vancouver, BC, Canada) using a PureLink® Genomic DNA Minikit (Invitrogen). The target amplicon DNA containing the desired CRISPR cut site was extracted using primers (Supplement Table 1), amplified with Q5® Hot Start 2× Master Mix (New England Biolabs, Ipswich, Massachusetts, US). Target DNA was purified with a PureLink® PCR cleanup kit (Invitrogen). 500 ng of target DNA was then added to PCR strip tubes, along with 1× NEB 3.1r buffer (New England Biolabs). Cas9/Cas12a preformed RNP was a positive control and AuNP samples at various stages were added at 5 ug Au core ± BME (Sigma-Aldrich), Vt = 50 μL. Samples were vortexed before incubation at 37 °C for 15 min. Samples then had 1 μL Proteinase K and 1 μL RNase A added (both from Invitrogen) and were incubated for 10 min at 28 °C, then 10 min at 32 °C before being heated to 95 °C to denature and cooled to 23 °C over 15 min. Resulting DNA samples were resolved for size on TapeStation (Agilent, Santa Clara, California, US) for quantification and 2% agarose gel electrophoresis for visualization.
5
Protease-Activated RNA Polymerase Assay
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All plasmids were constructed by Gibson Assembly 2× Master Mix (NEB); all PCR products were generated using Q5 Hot Start 2× Master Mix (NEB). E. coli strain S103030 (link) were transformed by electroporation with three plasmids: (i) a complementary plasmid (CP) that constitutively expresses a PA-RNAP with one of the three protease cut sites (Supplementary Fig. 2 ), (ii) an accessory plasmid (AP, Supplementary Fig. 4 ) that encodes gIII-luciferase (translationally coupled) under control of the T7 promoter, and (iii) an arabinose-inducible expression plasmid for one of the three proteases (EP, Supplementary Fig. 3 ). The HRV protease gene was purchased as IDT gblocks and cloned into the expression vector. The MBP-TEV fusion protein was amplified by PCR from pRK79341 (link). The MBP fusion was necessary for expression and solubility. We deployed a constitutively active HCV protease construct that includes the NS4a cofactor peptide42 (link). Cells were grown in 2xYT media to saturation in the presence of antibiotics and 1 mM glucose, then inoculated into 1 mL fresh media containing 1 mM glucose and antibiotics in a 96 well culture plate. After 4.5 h, 150 µL of the cultures were transferred to a black-wall clear-bottom assay plate and luciferase and OD600 measurements were taken using a Tecan Infinite Pro plate. The luminescence data was normalized to cell density by dividing by OD600.
6
SNP-Containing Enhancer Validation Assay
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Twenty-one regions containing at least one SNP that had an allele in a significant enhancer in either HT22 and/or 3T3-L1 cells were chosen for validation. These regions were PCRed from genomic DNA using Q5 Hot Start 2× Master Mix (NEB #M0494S) and were designed to be ~1 kb in size. Each region was cloned into the pGL4.23 luciferase vector containing firefly Luciferase and was tested for luciferase activity via co-transfection with renilla luciferase at a ratio of (1:50) in both 3T3-L1 cells and HT22 cells. Alleles were determined through Sanger sequencing. Renilla and firefly luciferase fluorescence was measured on a Promega GloMax microplate reader using the Dual-Luciferase Reporter Assay System (Promega #E1910). Firefly luciferase measurements were normalized to renilla measurements and then fold change over a control DNA region was calculated to determine enhancer activity.
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