All plasmid construction was performed using NEB HiFi assembly conceptually based on Gibson assembly (Gibson et al., 2009 (link)) using E. coli TOP10F as the initial plasmid propagating host. The primers used in this study are listed in Table 2 . Inducible promoter, Pm (3-methyl benzoate) was cloned from plasmid 138475 (pORTMAGE-Pa1) purchased form Addgene. The Pm promoter (1.946 kb) fragment was PCR amplified using primers Pm fragment forward/reverse (Table 2 ) and the vector pCAH01_emGFP was linearized by PCR amplification using primers pCAH01_emGFP vector forward/reverse (Table 2 ), the PCR amplicons of promoter and linearized vector were assembled using NEB HiFi assembly kit, resulting in pSGDA1. Similarly, sucrose phosphate synthase promoter (Psps; 792 bp) was PCR amplified using primers Psps fragment forward/reverse (Table 2 ) with template pDA21 (Table 1 ), vector pCAH01_emGFP was linearized using primers Psps vector forward/reverse (Table 2 ). Vector and insert were assembled using HiFi assembly (NEB) to construct pSGDA2.
Hifi assembly
Manufactured by New England Biolabs
Sourced in United States
The HiFi assembly is a DNA assembly method that can be used to combine multiple DNA fragments in a single reaction. It enables the seamless and efficient assembly of DNA constructs from individual parts.
Automatically generated - may contain errors
Lab products found in correlation
Q5 polymerase, by New England Biolabs (2 mentions)
Plvx tre3g ires, by Takara Bio (2 mentions)
Monarch pcr purification kit, by New England Biolabs (2 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (2 mentions)
Nucleospin plasmid mini kit, by Macherey-Nagel (2 mentions)
Moflo astrio, by Beckman Coulter (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Hifi assembly kit, by New England Biolabs (1 mentions)
Hifi dna assembly kit, by New England Biolabs (1 mentions)
Phusion hf dna polymerase, by New England Biolabs (1 mentions)
Pjet1.2 cloning vector, by Thermo Fisher Scientific (1 mentions)
5 3 race kit, by Roche (1 mentions)
Zero blunt topo vector, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Pcw57, by Addgene (1 mentions)
Plenticas9 blast, by Addgene (1 mentions)
Wizard genomic dna extraction kit, by Promega (1 mentions)
Kpni hf, by New England Biolabs (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
56 protocols using hifi assembly
1
Plasmid Construction Using NEB HiFi Assembly
2
Reconstitution of Centromeric Chromatin Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
) were generated using a HiFi DNA assembly kit (New England Biolabs).
For CENP-HIK Head expression, DNA encoding CENP-I 1-280, CENP-H 204-247, and CENP-K 165-269 were cloned, in that order, as a single polycistron into pRsfDuet using HiFi assembly (New England Biolabs), with a 3C-SNAP-2xStrepII tag in frame with CENP-I, thereby yielding the pRsfDuet CENP-HIK Head expression vector.
The N-terminal fragment of CENP-N (residues 1-212) was cloned into pGex6P-1 in-frame with an N-terminal 3C-cleavable GST tag using HiFi assembly (New England Biolabs, USA).
Human cDNAs comprising CENP-A and H4 were cloned by restriction-ligation into ORF1 and ORF2 of pRsfDuet. A bicistronic cassette encompassing 6xHis3C-tagged H2A and untagged H2B was cloned by restriction-ligation into pAcycDuet.
The coding regions of histone H2A, H2B, H4 and H3 were amplified by PCR from cDNA and cloned into pET28 plasmid. A double Strep-II tag together with a TEV cleavage site was attached to the N-termini of H3 and H2A proteins. For H3 octamer reconstitution, the histones H2A, H2B, H3 and H4 were assembled into a single pET28 plasmid by the USER methodology.
For CENP-HIK Head expression, DNA encoding CENP-I 1-280, CENP-H 204-247, and CENP-K 165-269 were cloned, in that order, as a single polycistron into pRsfDuet using HiFi assembly (New England Biolabs), with a 3C-SNAP-2xStrepII tag in frame with CENP-I, thereby yielding the pRsfDuet CENP-HIK Head expression vector.
The N-terminal fragment of CENP-N (residues 1-212) was cloned into pGex6P-1 in-frame with an N-terminal 3C-cleavable GST tag using HiFi assembly (New England Biolabs, USA).
Human cDNAs comprising CENP-A and H4 were cloned by restriction-ligation into ORF1 and ORF2 of pRsfDuet. A bicistronic cassette encompassing 6xHis3C-tagged H2A and untagged H2B was cloned by restriction-ligation into pAcycDuet.
The coding regions of histone H2A, H2B, H4 and H3 were amplified by PCR from cDNA and cloned into pET28 plasmid. A double Strep-II tag together with a TEV cleavage site was attached to the N-termini of H3 and H2A proteins. For H3 octamer reconstitution, the histones H2A, H2B, H3 and H4 were assembled into a single pET28 plasmid by the USER methodology.
3
Recombinant Dynein-2 Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A plasmid for baculovirus-mediated expression of the dynein-2 heavy chain (DYNC2H1), two intermediate chains (WDR60/DYNC2I1 and WDR34/DYNC2I2), light intermediate chain (DYNC2LI1), and single isoforms of the four dynein-2 light chains (DYNLRB1, DYNLL1, DYNLT1, and DYNLT2B/TCTEX1D2) was generated by removing redundant light chain isoforms (DYNLRB2, DYNLL2, DYNLT3, and LC8-like) from Addgene plasmid #132536 (Toropova et al, 2019 (link)) using restriction enzymes and HiFi assembly (NEB). The dynein-2 complex was purified from sf9 insect cells as described (Toropova et al, 2017 (link), 2019 (link)).
4
Optimized Kinase Translocation Reporter Plasmid
5
Heterologous Expression of Pf2826 in U. maydis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To express the P. flocculosa effector Pf2826 in U. maydis strain FB1, we followed the cloning and transformation procedures outlined in Teichmann et al. [28 (link)]. Briefly, the strong constitutive Potef promoter was used to express Pf2826 and a carboxin resistance (ip) cassette as a selectable marker in U. maydis. Coding sequence of Pf2826 was amplified and introduced into p123 under the control of the strong Potef promoter using NEB Hi-Fi assembly following the manufacturer’s instructions (NEB, USA). Prior to integrative transformation into the ip locus of U. maydis strain FB1, plasmids were linearized with SspI. For selection of transformants, PDA plates containing 2 µg/ml carboxin were used. Mutants were confirmed by PCR amplification and validated by sequencing. Assessment of the biocontrol activity of U. maydis wild-type strain FB1 and two independent FB1 transformants overexpressing Pf2826 strains was performed exactly like P. flocculosa tripartite assay.
6
Construction of Cyanobacterial Reporter Plasmid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate the vectors to test the expression of
the reporter gene (eyfp), a DNA fragment containing
a cpcBA promoter from Synechocystis sp. PCC 6803 (PcpcBA6803), a His10-tag, and an eyfp gene was
ordered from a gene-synthesis service (Genomics, New Taipei City,
Taiwan) and subcloned into a pJET1.2 cloning vector (Thermo Fisher,
Waltham, MA). A transcriptional terminator from the rrnB gene (TrrnB) of E. coli strain DH5α was amplified by PCR and introduced downstream
of the eyfp gene through HiFi assembly (New England
Biolabs, Ipswich, MA). The resulting plasmid was named pSWYR (SW itch-eY fp-TR rnB). In pSWYR, the PcpcBA6803 is flanked by restriction
sites for EcoRI/HindIII and NcoI and can be replaced with other promoters
cloned from the genomic DNA of Syn7335 and Leptolyngbya sp. JSC-1 for activity screening. The promoter sequence of PchlF7335 was cloned into pSWYR
through HiFi assembly to replace PcpcBA6803. The DNA fragment containing a promoter, eyfp, and TrrnB was digested from pSWYR by
XhoI and BamHI or by HindIII and XhoI and subcloned into plasmids
pRL1342Em and pRL1342Km for conjugal delivery to target cyanobacteria.
All PCR amplification for cloning was performed with Phusion HF DNA
polymerase (New England Biolabs, Ipswich, MA). SeeTable S2 for the details of primers used for PCR.
the reporter gene (eyfp), a DNA fragment containing
a cpcBA promoter from Synechocystis sp. PCC 6803 (PcpcBA6803), a His10-tag, and an eyfp gene was
ordered from a gene-synthesis service (Genomics, New Taipei City,
Taiwan) and subcloned into a pJET1.2 cloning vector (Thermo Fisher,
Waltham, MA). A transcriptional terminator from the rrnB gene (TrrnB) of E. coli strain DH5α was amplified by PCR and introduced downstream
of the eyfp gene through HiFi assembly (New England
Biolabs, Ipswich, MA). The resulting plasmid was named pSWYR (
sites for EcoRI/HindIII and NcoI and can be replaced with other promoters
cloned from the genomic DNA of Syn7335 and Leptolyngbya sp. JSC-1 for activity screening. The promoter sequence of PchlF7335 was cloned into pSWYR
through HiFi assembly to replace PcpcBA6803. The DNA fragment containing a promoter, eyfp, and TrrnB was digested from pSWYR by
XhoI and BamHI or by HindIII and XhoI and subcloned into plasmids
pRL1342Em and pRL1342Km for conjugal delivery to target cyanobacteria.
All PCR amplification for cloning was performed with Phusion HF DNA
polymerase (New England Biolabs, Ipswich, MA). See
7
Generating Episomal BDF5 Mutants in L. mexicana
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate episomal addbacks the BDF5 CDS was amplified from L. mexicana genomic DNA and cloned into the pNUS C-Ter GFP NEO (pGL1132) using HiFi Assembly (NEB) to generate a complementation vector. This was used as a base for site-directed mutagenesis using the Q5 Mutagenesis product (NEB) to generate mutations in the conserved asparagine residues N90 (OL9577 and OL10352), N257 (OL9579 and OL10353) to phenylalanine in BDF5 BD5.1 and BD5.2 (Supplementary Data 1 ). Log-phase promastigotes were transfected with 2–5 µg plasmid DNA and maintained as population under G418 selection.
8
Constructing Plasmids via Multiple Assembly Methods
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids were constructed using golden-gate assembly (47 (link), 48 (link)), HiFi assembly (New England Biolabs), and yeast recombination (49 (link)), as described in SI Appendix, Supplementary Materials and Methods . All inserts were confirmed by Sanger sequencing. Plasmids were chemically transformed into E. coli strains (50 (link)) and mobilized into rhizobia via biparental conjugation with E. coli ST18 (51 (link)) or via triparental conjugation with a helper E. coli strain carrying plasmid pRK2013. Plasmids with R6K replicons were maintained at low copy number in E. coli EC100D Pir+ where possible.
9
Transgenic Arabidopsis Line Construction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding sequences of MtIPD3, S50D-IPD3, and IPD3-Min (Genbank EF569224.1 ; Yano et al. 2008 (link), Singh et al 2014 (link)) were synthesized (Integrated DNA Technologies, Research Triangle Park, NC) and assembled by Hi-Fi assembly (New England Biolabs, Ipswich, MA) into a modified pCAMBIA0380 expression construct (Genbank AF234290.1 ) under control of the Arabidopsis Ubiquitin 10 promoter (Ivanov and Harrison 2014 (link)). 35S:mCherry amplified from pC-GW-mCherry (Genbank KP826771.1 ) (Dalal et al. 2015 (link)) was the selection marker. Arabidopsis were transformed as described by Davis et al. (2009) (link). Seeds were screened by fluorescence and PCR during segregation and lines were brought to homozygosity.
10
Molecular Cloning and Genetic Manipulation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacteria used in this study (Table S1 ) were cultured in TY (Beringer, 1974 ) or UMS (Brown & Dilworth, 1975 ; Haskett, Paramasivan, et al., 2022b ; Poole et al., 1994 (link)). Plasmids (Table S2 and Data S2 ) were constructed using HiFi assembly (New England Biolabs) or BEVA modular golden‐gate assembly (Geddes, Mendoza‐Suárez, & Poole, 2019a (link); Weber et al., 2011 (link)) and were mobilized into strains of interest via di‐parental mating with E. coli ST18 (Thoma & Schobert, 2009 (link)). For mini‐Tn7 integration into the chromosome of AcLP, tri‐parental matings were required to additionally mobilize the transposase helper plasmid pTNS3 (Choi & Schweizer, 2006 (link)). 5′‐RACE was performed using a 2nd Generation Roche 5′/3′ RACE Kit as per the manufacturer's recommendations.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!