The human codon optimized LtrA sequence was generated from overlapping oligonucledotides by assembly PCR [65 (link)]. Oligonucleotides containing hLtrA sequence were synthesized by HHMI/Keck Oligonucleotide Synthesis Facility (Yale) and PCR reactions were carried out by using Vent DNA polymerase (New England Biolabs), high annealing temperatures (58–60°C), and manual hot start–i.e., adding Vent DNA polymerase after sample temperature reached 94°C). PCR products were gel-purified and digested with EcoRI and XbaI or HindIII and XbaI, then cloned into pKSBluescript (Agilent) to form pKS-hLtrA and confirmed by sequencing. The assembled ORF was re-cloned into a pIRES vector (Clontech) to generate phLtrA1.
Vent dna polymerase
Manufactured by New England Biolabs
Sourced in United States, United Kingdom, Canada, Germany
Vent DNA polymerase is a thermostable DNA polymerase enzyme isolated from the hyperthermophilic archaeon Thermococcus litoralis. It exhibits 3'-5' exonuclease activity and is capable of synthesizing DNA at high temperatures.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (9 mentions)
Pcdna3, by Thermo Fisher Scientific (6 mentions)
Taq dna polymerase, by New England Biolabs (4 mentions)
Restriction enzyme, by New England Biolabs (3 mentions)
Phusion dna polymerase, by New England Biolabs (3 mentions)
Restriction enzyme, by Thermo Fisher Scientific (3 mentions)
Antarctic phosphatase, by New England Biolabs (3 mentions)
T4 pnk, by New England Biolabs (3 mentions)
Oligonucleotide, by Merck Group (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
T4 dna ligase, by Promega (3 mentions)
Restriction endonuclease, by New England Biolabs (2 mentions)
Calf intestine alkaline phosphatase, by New England Biolabs (2 mentions)
Taq dna polymerase, by Roche (2 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Nadph, by AppliChem (2 mentions)
Benzamidine sepharose, by GE Healthcare (2 mentions)
E coli er2566 competent cells, by New England Biolabs (2 mentions)
Gsh sepharose4 fast flow, by GE Healthcare (2 mentions)
65 protocols using vent dna polymerase
1
Cloning and Sequencing of codon-optimized LtrA
2
Radiolabeled Reagents for Molecular Biology
3
DNA Manipulation Protocol Optimization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All restriction and DNA modifying enzymes (T4 DNA ligase, Vent DNA polymerase, and calf intestine alkaline phosphatase) were from New England Biolabs (Ipswich, MA). Other chemicals were from sources recently described17 (link)23 (link).
4
Recombinant Protein Production and Cell Culture Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Restriction enzymes and Vent DNA polymerase were obtained from NEB. Human MMPs-1/2/7 were purchased from AnaSpec. The catalytic domain of human MMP-14 (cdMMP-14) was produced and biotinylated as previously described [31 (link)]. The following vectors and cell lines were from lab stock: periplasmic expression vector pMopac16 [52 (link)], pBAD33 [53 (link)], DsbA/C chaperon plasmids [54 (link)] pBAD-DsbA, pBAD-DsbC, pBAD-AC1P (both DsbA and DsbC under one PBAD promoter) and pBAD-AC2P (DsbA and DsbC under two separated PBAD promoters), and E. coli strains Jude-I [DH10B F’::Tn10(Tetr)] and BL21(DE3). A human mammary basal/claudin low carcinoma cell line (MDA-MB- 231, ATCC) was cultured in Dulbecco’s Modified Eagles Medium (DMEM) (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin (pen-strep) (Gibco) at 37 °C in 5% CO2. Imaging media for MDA-MB-231 cells was the same as the subculturing media, with the exception that no phenol red was included and that 12 mM HEPES (Sigma-Aldrich) was included.
5
Generating Cysteine Mutants of TcTIM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA sequence U53867 for TcTIM at the NCBI database was used to create single mutants. All mutants except the C15A mutant were constructed on a pET-HisTEVP-modified plasmid45 (link) using the QuickChange protocol (Agilent Technologies, CA). The C15A mutant was constructed using the plasmid pET-3a containing the WT TcTIM sequence as a template and introducing the mutation with the polymerase chain reaction (PCR) using Vent DNA Polymerase (New England Biolabs, MA, USA). The external nucleotides were the T7 Promoter and the T7 Terminator (Novagen). The mutation was introduced as follows: 25 cycles for 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C and the extension incubation for 10 min at 72 °C. The PCR product was cloned into the pET-3a expression vector (Novagen) as an NdeI-BamHI fragment.
The mutagenic oligonucleotides used to produce TcTIM Cys-mutants are shown inSupplementary Table S6 . The plasmids containing the different mutants were transformed into the Escherichia coli BL21 Codon Plus (DE3)-RIL strain (Agilent Technologies, CA).
The mutagenic oligonucleotides used to produce TcTIM Cys-mutants are shown in
6
Plasmid Cloning Using Standard Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid cloning was performed primarily using standard PCR and restriction enzyme cloning with Vent DNA polymerase (New England Biolabs (NEB)), Taq DNA Polymerase (NEB), Phusion DNA Polymerase (NEB), restriction enzymes (NEB; Thermo Fisher), T4 DNA Ligase (NEB), Antarctic Phosphatase (NEB) and T4 PNK (NEB). Golden gate assembly and Gibson assembly were also utilized. The pBI-EYFP reporter was described previously (Addgene #58855) (25 (link)). GBP2-containing and GBP7-containing source plasmids were a generous gift from Constance Cepko (31 (link)). Plasmids were transformed into chemically competent TOP10 Escherichia coli (Thermo Fisher), and cells were grown at 37°C.
7
Purification of E. coli NusG Variants
8
Directed Evolution of AspBHI Enzyme
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All enzymes, plasmids and bacterial strains, if not otherwise specified, were obtained from New England Biolabs (NEB). Escherichia coli codon optimized AspBHI with an N-terminal 6xHis tag was cloned into a pUC19 derivative pZZ1 (Z. Zhu, NEB) between NdeI and BamHI sites4 (link). Site-directed mutagenesis was carried out by inverse PCR using Vent® DNA polymerase and mutagenic primers designed with NEB in-house software. The entire alleles in AspBHI variants were sequenced to confirm the desired mutation.
9
Generation of Mutant α-Syn-GFP Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type α-syn-GFP plasmid was obtained from Origene (RG210606; Rockville, MD). α-syn-GFP mutants were created by site-directed mutagenesis with the following primers (Invitrogen, Life Technologies, Burlington, Canada): for N122S (PVDPDS): fwd: 5′-GTGGATCCTGACTCTGAGGCTTATG-3′; rev: 5′- CATAAGCCTCAGAGTCAGGATCCAC-3′; for D121S (PVDPSN): fwd: 5′- CCTGTGGATCCTTCTAATGAGGCTTATG-3′; rev: 5′- CATAAGCCTCATTAGAAGGATCCACAGG-3′.
Briefly, wild-type α-syn-GFP was amplified in the presence of either primer pair using Vent DNA polymerase (M0254L; New England Biolabs, Ipswich, MA) to produce a blunt-ended product. Resultant PCR products were digested with DpnI restriction enzyme (New England Biolabs) to remove parental methylated DNA. The remaining DNA was purified, ligated, and transformed into NovaBlue Singles competent cells (EMD Millipore, Billerica, MA). Plasmids obtained from ampicillin-resistant clones were sequenced by StemCore Laboratories (Ottawa, Canada) using a primer complementary to the CMV promoter (5′-AAA TGG GCG GTA GGC GTG-3′).
LMP1 expression vector (p1990 SV40-LMP1) was created by Bill Sugden and obtained from Addgene (26654; Cambridge, MA).
Briefly, wild-type α-syn-GFP was amplified in the presence of either primer pair using Vent DNA polymerase (M0254L; New England Biolabs, Ipswich, MA) to produce a blunt-ended product. Resultant PCR products were digested with DpnI restriction enzyme (New England Biolabs) to remove parental methylated DNA. The remaining DNA was purified, ligated, and transformed into NovaBlue Singles competent cells (EMD Millipore, Billerica, MA). Plasmids obtained from ampicillin-resistant clones were sequenced by StemCore Laboratories (Ottawa, Canada) using a primer complementary to the CMV promoter (5′-AAA TGG GCG GTA GGC GTG-3′).
LMP1 expression vector (p1990 SV40-LMP1) was created by Bill Sugden and obtained from Addgene (26654; Cambridge, MA).
10
Molecular Cloning Techniques Detailed
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids employed in this study are listed in Table A of S1 Text . PCRs were performed with the Taq DNA polymerase (Roche, NZyTech) for standard amplifications in screenings or with the proof-reading DNA polymerases Herculase II Fusion (Agilent Technologies) or Vent DNA polymerase (NEB) for cloning purposes. When indicated, DNA was synthesized by GeneArt (Life Technologies). All DNA constructs were confirmed by DNA sequencing (Secugen and Macrogen). Oligonucleotides used in this work were obtained from Sigma and are described in Table C of S1 Text .
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!