Plasmids (listed in Supplementary Data 2 ) were constructed by PCR-based uracil-specific excision reagent (USER) cloning method63 (link),64 (link) or using Gibson cloning65 (link). For USER cloning, one microliter of 5x HF buffer (Thermo Scientific) and 1U of USER™ enzyme mix (New England Biolabs, 1 U/µl) were added to 8 µl of the mixture of purified PCR products, plasmid-backbone or genes. All PCR products were amplified with oligonucleotides (Integrated DNA Technologies) having uracil incorporated, utilizing Phusion U polymerase (Thermo Fisher Scientific). The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. Eight microliters MilliQ was added to the reactions, reaching a final volume of 20 µl. In all, 2.5 µl of the diluted USER reaction was used to transform 50 µl competent E. coli. Gibson assemblies were carried out using Gibson Assembly Cloning Kit (NEB). Fragments (motif cassette) with ∼30 bp overlap regions were mixed together with the backbone fragment in 1:1 molar ratio together with an equal volume of Gibson master mix, incubated at 50 °C for 1 h and immediately transformed into E. coli.
Phusion u polymerase
Manufactured by Thermo Fisher Scientific
Phusion U polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It exhibits robust performance and exhibits 3' to 5' exonuclease proofreading activity, resulting in low error rates during PCR.
Automatically generated - may contain errors
Lab products found in correlation
User enzyme mix, by New England Biolabs (2 mentions)
Clonejet vector, by Thermo Fisher Scientific (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Hf buffer, by Thermo Fisher Scientific (2 mentions)
Oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Gibson assembly cloning kit, by New England Biolabs (1 mentions)
Cutsmart, by New England Biolabs (1 mentions)
Nb bsmi, by New England Biolabs (1 mentions)
User enzyme, by New England Biolabs (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
E coli top10 cells, by Thermo Fisher Scientific (1 mentions)
Geneart strings dna fragment, by Thermo Fisher Scientific (1 mentions)
5 aza dc, by Merck Group (1 mentions)
Qubit high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
10 protocols using phusion u polymerase
1
Plasmid Construction Using USER and Gibson
2
Plasmid Cloning Using USER Method
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All plasmids in this study were cloned
using uracil specific excision reagent (USER) cloning (New England
Biolabs) and the EasyClone method.27 (link) Genetic
parts for assembly into plasmids and USER vector plasmids were amplified
using PhusionU polymerase (Thermo Fisher Scientific). Plasmids containing
the GPCRs had a Kozak sequence (AAAACA) in front of the start codon
of the receptor. Synthetic genes were ordered from TWIST Bioscience,
and custom oligos were ordered from IDT or used from previous publications.28 (link) The complete list of all gBlocks and plasmids
and yeast strains can be found inSupporting Information Tables S6–S8. The plasmids were transformed into the chemically
competent DH5α strain by heat-shocking for 45 s at 42 °C
and recovered on Luria–Bertani plates supplemented with 100
μg/mL ampicillin.
using uracil specific excision reagent (USER) cloning (New England
Biolabs) and the EasyClone method.27 (link) Genetic
parts for assembly into plasmids and USER vector plasmids were amplified
using PhusionU polymerase (Thermo Fisher Scientific). Plasmids containing
the GPCRs had a Kozak sequence (AAAACA) in front of the start codon
of the receptor. Synthetic genes were ordered from TWIST Bioscience,
and custom oligos were ordered from IDT or used from previous publications.28 (link) The complete list of all gBlocks and plasmids
and yeast strains can be found in
competent DH5α strain by heat-shocking for 45 s at 42 °C
and recovered on Luria–Bertani plates supplemented with 100
μg/mL ampicillin.
3
USER Cloning of BioBricks into Vectors
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All plasmids and BioBricks used are listed in Supplementary Tables 2, 3 , respectively. BioBricks were amplified by PCR using Phusion U polymerase (Thermo Fisher Scientific) with following thermal program: 98°C for 30 s, 30 cycles of (98°C for 10 s, 51°C for 30 s, 72°C for 30 s/kb), and 72°C for 5 min. BioBricks were then resolved on 1% agarose gels and purified using NucleoSpin®Gel and PCR Clean-up kit (Macherey-Nagel). The assembly of BioBricks into vectors was conducted by USER cloning (Holkenbrink et al., 2017 (link)). The parental vectors were digested with FastDigest SfaAl (Thermo Fisher Scientific) at 37°C for 60 min and nicked with Nb.BsmI (New England BioLabs) at 65°C for 60 min. BioBricks with compatible overhangs and SfaAl/Nb.BsmI-treated parental vectors prepared as above were incubated in CutSmart® buffer with USER enzyme (New England BioLabs) for 25 min at 37°C, followed by 10 min at 25°C, and transformation into E. coli. The colonies were tested by colony PCR and correct assembly was verified by DNA sequencing.
4
Rapid Plasmid Construction via USER Cloning
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Primers used in this study are listed in Table 2 . All fragments were amplified with oligomers having uracil incorporated, using the Phusion U polymerase (Thermo Scientific). The plasmids and promoter library were constructed by the uracil-specific excision reagent (USER) cloning method (Geu-Flores et al. 2007 (link); Nour-Eldin et al. 2006 (link)). In brief, 1 μl of 5× HF buffer (Thermo Scientific) and 1 U of USER™ enzyme mix (New England Biolabs, 1 U/ml) were added to 10 µl of the mixture of purified PCR products, plasmid backbone, or genes.
The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. 8 µl of water was added to the reactions, reaching a final volume of 20 µl. 5 µl diluted USER mixture was used to transform chemically competent E. coli TOP10 cells (Thermo Scientific) (Sambrook and Russell 2001 ).
The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. 8 µl of water was added to the reactions, reaching a final volume of 20 µl. 5 µl diluted USER mixture was used to transform chemically competent E. coli TOP10 cells (Thermo Scientific) (Sambrook and Russell 2001 ).
5
Plasmid Assembly and Yeast Transformation
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All plasmids used in this study were assembled by USER™ (uracil-specific excision reagent) cloning (New England Biolabs). Biobricks used to assemble the plasmids were amplified by PCR using PhusionU polymerase (Thermo Fisher Scientific). S. cerevisiae strains were transformed by the lithium acetate/single-stranded carrier DNA/PEG method previously described [13 (link)]. The complete list of strains and plasmids used in this study is provided in Supplementary Tables S1 and S2 .
6
Bisulfite PCR and Sequencing Protocol
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Up to 2 µg genomic DNA, or the total amount to eluted material from ChIP, was used for bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen). Bisulfite PCR was carried out using the PhusionU polymerase (Thermo Fisher Scientific) with the primers indicated in Supplementary Table 1 using the following conditions: initial denaturation at 95 °C for 5 min, followed by 45 cycles of 1 min at 95 °C, 1 min at 50–60 °C (dependent on the primer pair) and 1 min at 72 °C, followed by 5 min of final extension at 72 °C. Amplicons were cloned into the CloneJET vector (Thermo Fisher Scientific), sequenced by Sanger sequencing and analyzed using QUMA63 (link).
7
Bisulfite Sequencing of Genomic DNA
8
Heterologous Expression of Fungal ABA Biosynthesis
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The plasmids, biobricks and primers used in this study are listed in Tables S2, S3 and S4 (Supporting Information), respectively. Phusion U polymerase (Thermo Scientific) was used to amplify the biobricks with polymerase chain reactions (PCR), which were assembled into EasyCloneYALI plasmids by Uracil-Specific Excision Reagent (USER) cloning (Holkenbrink et al. 2018 ). The USER reactions were transformed into E. coli and correct assembly was verified by sequencing. The genes encoding BcABA1 (NCBI reference: XP_024550391.1), BcABA2 (NCBI reference: XP_024550390.1), BcABA3 (NCBI reference: XP_024550392.1), BcABA4 (NCBI reference: XP_001553969.2), BcCPR1 (NCBI reference: XP_001558194.1), a putative B. cinerea α-ionylideneethane synthase BcABA5 (amino sequence as from strain ATCC58025 described in Izquierdo-Bueno et al. 2018 (link)), and the Arabidopsis thaliana ABA transporters AtDTX50 and AtABCG25 (UniProt references: Q9FJ87 and Q84TH5, respectively) were ordered as GeneArt Strings DNA fragments from Thermo Fischer Scientific. The DNA sequences were codon-optimized for Y. lipolytica using GeneArt standard algorithm. The DNA sequences are provided in the Supporting Information.
9
Methylation analysis of p16 promoter
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Colony-derived SHE cultures were analysed for patterns of methylation at the p16 promoter region. gDNA was bisulphite converted prior to methylation analysis. A 457 bp region containing the SH p16 promoter was then amplified by PCR using Phusion U polymerase (Thermo Scientific) and gel-excised before insertion into pJET1.2/blunt linearised cloning vector (Thermo Scientific). DNA was extracted from a minimum of 10 bacterial cultures containing the correctly ligated plasmid and bisulphite sequenced to estimate the extent of methylation present at each CpG site. Four MT colony-derived cell lines (BP MT2, BP MT6, BP MT7 and BP MT8) were treated with the demethylating agent 5′-Aza-2′-deoxycytidine and monitored over a period of 4 weeks. Two doses of 5 μM 5-aza-dC (Sigma) were added to fresh culture media at 4 h intervals before removal.
10
Genome-wide PCR Amplification and Sequencing
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Genomic DNA was isolated as described above. Following genomic DNA isolation, 1 μL of the isolated DNA (1–10 ng) was used as input for the first of two PCR reactions. Genomic loci were amplified in PCR1 using PhusionU polymerase (Thermo Fisher Scientific). PCR1 primers for genomic loci are listed in Table S2 under the HTS_fwd and HTS_rev columns. PCR1 was performed as follows: 95 °C for 3 min; 30–35 cycles of 95 °C for 15 s, 61 °C for 20 s, and 72 °C for 30s; 72°C for 1 min. PCR1 products were confirmed on a 1% agarose gel. 1 μL of PCR1 was used as an input for PCR2 to install Illumina barcodes. PCR2 was conducted for nine cycles of amplification using a Phusion HS II kit (Life Technologies). Following PCR2, samples were pooled and gel purified in a 1% agarose gel using a Qiaquick Gel Extraction Kit (Qiagen). Library concentration was quantified using the Qubit High-Sensitivity Assay Kit (Thermo Fisher Scientific). Samples were sequenced on an Illumina MiSeq instrument (paired-end read, read 1: 200–280 cycles, read 2: 0 cycles) using an Illumina MiSeq 300 v2 Kit (Illumina).
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