TIR-MAPT transcript was obtained from an SH-SH5Y RNA extract, using specific oligos (TauNt and TauD, see Supplementary table 2, online resource) and was cloned into pBlueScript-SK + (212,205, Agilent Technologies), thanks to a TA-cloning strategy [37 (link)]. Using specific oligos that include the appropriate restriction sites (A1 and TIR-T-BglII and ET-T-BglII, see Supplementary table 2, online resource) TIR-MAPT and ET-MAPT were subcloned into a eukaryotic expression vector pSG5 (216201, Agilent technologies). Similarly, Tau isoforms were cloned into a prokaryotic expression vector pRK172 using specific oligos (TAU-PRK172 fw, TIR-T-pRKpWPI rv and ET-T-pRKpWPI rv; see Supplementary table 2, online resource). After cloning, all vectors were sequenced using the described oligos (Supplementary table 2, online resource).
Pbluescript sk
Manufactured by Agilent Technologies
Sourced in United States
PBluescript SK+ is a plasmid cloning vector used for DNA cloning and sequencing. It contains a multiple cloning site, a lacZ gene for blue-white screening, and an ampicillin resistance gene for selection. The plasmid has a pUC origin of replication, allowing for high-copy number propagation in Escherichia coli.
Automatically generated - may contain errors
Lab products found in correlation
Maxiprep kit, by Macherey-Nagel (2 mentions)
E coli xl1 blue cells, by Agilent Technologies (2 mentions)
Pbluescript, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Bamhi, by New England Biolabs (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Anti dig ap antibody, by Roche (1 mentions)
Bm purple ap substrate, by Merck Group (1 mentions)
12 protocols using pbluescript sk
1
Cloning and Sequencing of MAPT Isoforms
2
Preparation and Manipulation of pSG483 Plasmid
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Negatively supercoiled pSG483 (2927 bp), a pBlueScript SK+ (Agilent) derivative containing a single Nb.BbvCI site, was prepared from E. coli XL-1 blue cells (Agilent) using a maxiprep kit (Macherey-Nagel). A portion of this sample was treated with BamHI to form linear plasmid. Another portion of this sample was nicked with Nb.BbvCI and an aliquot was removed to make a nicked pSG483 stock. The kDNA substrate was purchased from Inspiralis; restriction enzymes were from NEB.
3
Luciferase Assay for Transcriptional Regulation
4
High-Copy Cas9 Vector Generation
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To generate a high-copy number version of the Cas9 vector for injection, a NotI fragment containing Cas9 and the hsp70 regulatory regions was excised from phsp70-Cas9 (Gratz et al. 2013a (link)). This fragment was ligated into the NotI site of pBluescript SK+ (Agilent Technologies).
5
Unconstrained DNA Molecules for Torque Measurements
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A 7.9 kb-long random-sequence DNA molecule lacking any nucleosome-positioning sequences nor alpha-satellite DNA sequence was made by taking a fragment of the pBlueScript-1,2,4+pSfv1 plasmid, which consists of fragments of Lambda DNA and a fragment from pSfv1 (Invitrogen) in pBlueScript SK+ (Agilent). Alpha satellite DNA sequence plasmids were obtained as described in [43 (link)]. From this plasmid, DNA fragments each containing 5 alpha satellite sequence repeats were taken and self-ligated to each other to make a final construct of 3.4 kb DNA as a second substrate. For surface immobilization, a 500 bp-long DNA fragment labeled with multiple Dibenzocyclooctyne (DBCO) molecules was ligated at one end of the DNA molecules, while the other end was ligated with a 500 bp-long DNA fragment labeled with multiple biotin molecules for magnetic-bead attachment. In the experiments, we selected, by measuring their torque-responses, rotationally unconstrained DNA molecules that can freely rotate due to the presence of a nick. The complete DNA sequences are given in S1 Text
6
Preparation and Manipulation of pSG483 Plasmid
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Negatively supercoiled pSG483 (2927 bp), a pBlueScript SK+ (Agilent) derivative containing an Nb.BbvCI site, was prepared from E. coli XL-1 blue cells (Agilent) using a maxiprep kit (Macherey-Nagel). A portion of this sample was treated with BamHI (NEB) to form linear plasmid. Another portion of this sample was nicked with Nb.BbvCI (NEB) and an aliquot was removed to make a nicked pSG483 stock. A further aliquot was ligated with T4 DNA ligase (NEB), to form relaxed pSG483. All plasmid forms were purified by phenol/chloroform extraction and ethanol precipitation prior to use.
7
Production of MCF10A Cell Lines
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Production of MCF10A control and shEIF3E cell lines has been previously described.27 MCF10A shCtrl and shEIF3E cell lines were transfected in 100 mm tissue culture plates with 5 μg of DNA at a ratio of 1:50 of a T7‐tagged PRP4K expression vector16 or pBluescript SK‐ (Stratagene/Agilent Technologies, Santa Clara, CA, USA) using the Neon Transfection System (Thermo) according to the manufacturer's directions. A pulse voltage of 1400V, pulse width of 20 ms, and pulse number of 2 were used. Cells were harvested for RT‐qPCR, fixed and permeabilized for immunofluorescence 36 h post‐transfection.
8
Capped cRNA Synthesis and Mutagenesis
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Capped cRNA transcripts coding for hERG (NM_000238), hKv10.1 (NM_172362) and hKv1.3 (NM_002232) were obtained from linearized plasmids with the mMessage mMachine T7 transcription kit (Thermo-Fisher) following manufacturer's instructions. The concentrations were adjusted to 1 μg/μL for injection in Xenopus laevis oocytes. The S631C mutation in the sequence encoding hERG was introduced by overlap extension PCR. Briefly, hERG cDNA and the primers herg-008S (5′-CATGGCCCGGACGCGCTCCC-3′) and herg-018AS (5′-GAATTCGTGTTGGGGCAGACGTTGCCGAAGC-3′) or herg-019S (5′- CGTCTGCCCCAACACGAATTCAGAGAAGATC-3′) and herg-004AS (5′- CTAGTGCTGCAGCAGTGAGCGG-3′) were used to PCR amplify two fragments of 1110 bp and 334 bp. The two fragments were purified and subjected to a second round of PCR amplification to obtain a 1423 bp-long fragment that was cloned in pBluescript-SK (Agilent). The presence of the mutation was confirmed by sequencing the entire insert, and then exchanged with the corresponding fragment of the wild-type sequence.
9
HA-tagged PERV-C Design and Characterization
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Design of HA-tagged PERV-C was performed starting with the viral backbone of PERV-C(5683) (KY352351.2) [26 (link)] cloned in pBluescript SK (+) (Agilent Technologies, Germany). EnvC of PERV-C(5683) was structurally analyzed using the open source tool PSIPRED [59 (link)] to highlight sections of amino acids that do not bear structural motifs (Additional file 1 : Fig. S1A). Only sequence sections defined as coil were selected for integration of the tag. Integration into helixes or ß-strands was avoided. Based on sequence alignments, structural analysis and on published functional studies [11 (link), 26 (link), 53 (link), 60 (link)] the HA-tag was introduced at AA 44–52, AA 86–94 or AA 639–647 resulting in SP-HA, HA-VRA and RPep-HA, respectively. Corresponding sequences are shown in Additional file 1 : S1B–D.
Integration of the HA-tag into PERV-C(5683) was performed by site directed mutagenesis using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Germany) following the manufacturer’s instruction. Primers used are shown in Table1 .
Integration of the HA-tag into PERV-C(5683) was performed by site directed mutagenesis using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Germany) following the manufacturer’s instruction. Primers used are shown in Table
10
Transgenic Strain Construction in C. elegans
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PHA-1 plasmid (gift from Krastosis lab, University of Chicago) and pBluescript SK(-) (Agilent Technologies, USA) were used during construction of transgenic strains. PCR fragments containing promoters of choice, snb-1 or odr-2 and 9E, were generated using standard cloning protocols. psnb-1::odr-2 and snb-1::9E geneBlock fragments were obtained from IDT. pglr-4, psnb-1, 9E was cloned out of plasmids generated in house (pglr-4- Krastosis lab; psnb-1 and 9E- Krishnan lab). pacr-2 was cloned out of genomic DNA isolated from wild-type worms. All the PCR fragments had a unc-54–3′UTR sequence on their 3′ end for better expression in worms (Merritt et al., 2008 (link)).
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