Genomic DNA was extracted from cells originated patient’s bone marrow and iPS cells using QIAamp DNA Mini Kit (QIAGEN). The CEPF2-FGFR_ER primer pair used to amplify the CEP110-FGFR1 genomic fusion locus is described in S2 Table . To determine the fusion point, the PCR products ware cloned into pCR4Blunt-TOPO (Life Technologies as described by the manufacturer) and sequenced (S1 Fig .).
Pcr4blunt topo
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PCR4Blunt-TOPO is a linearized vector that enables the direct cloning of blunt-ended PCR products. It is designed for efficient and rapid cloning of PCR amplicons into the vector without the need for additional enzymatic processing steps.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
G 418 sulfate, by Fujifilm (1 mentions)
Pcdna3, by Promega (1 mentions)
Human liver cdna library, by Takara Bio (1 mentions)
Pcdh cmv mcs ef1 puro lentivector, by System Biosciences (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Mightyamp dna polymerase, by Takara Bio (1 mentions)
Fastap, by Thermo Fisher Scientific (1 mentions)
Mix go e coli transformation kit, by Zymo Research (1 mentions)
Kod hot start dna polymerase, by Merck Group (1 mentions)
Abi steponeplus, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
13 protocols using pcr4blunt topo
1
Genomic Fusion Identification from Bone Marrow and iPS Cells
2
Cloning and Expression of Rat and Human NPC1L1
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The cDNA encoding rat NPC1L1 (rNPC1L1) (GenBank AY437867) was generated by PCR amplification from rat jejunum single-strand cDNA (GenoStaff) and cloned into pCR4 Blunt TOPO (Life Technologies). The rNPC1L1 cDNA from this construct was inserted into the expression vector pcDNA3.1 (Life Technologies) and pCDH-CMV-MCS-EF1-Puro Lentivector (System Biosciences).
The cDNA encoding the human NPC1L1 (hNPC1L1) (GenBank AY437865) was generated by PCR amplification from a human liver cDNA library (Takara Bio) and cloned into pCR2.1 (Life Technologies). The hNPC1L1 cDNA was modified by inserting enhanced green fluorescent protein (EGFP) sequence at the C terminus, and then ligated into pcDNA3.1. Site-directed mutagenesis was performed by a PCR-based strategy using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories). The influenza virus hemagglutinin (HA) tag was inserted between S986 and L987 of hNPC1L1.
The pcDNA3.1 constructs encoding rNPC1L1, hNPC1L1-EGFP, hNPC1L1L1072T/L1168I-EGFP, HA-hNPC1L1-EGFP, or HA-hNPC1L1L1072T/L1168I-EGFP were transfected into HEK293 cells using FuGENE HD (Promega). Cells were selected by culturing in the presence of 1 mg/ml G418 sulfate (Wako Pure Chemical Industries). For FACS analysis, HEK293 cells stably expressing HA-hNPC1L1L1072T/L1168I-EGFP were cloned by limiting dilution.
The cDNA encoding the human NPC1L1 (hNPC1L1) (GenBank AY437865) was generated by PCR amplification from a human liver cDNA library (Takara Bio) and cloned into pCR2.1 (Life Technologies). The hNPC1L1 cDNA was modified by inserting enhanced green fluorescent protein (EGFP) sequence at the C terminus, and then ligated into pcDNA3.1. Site-directed mutagenesis was performed by a PCR-based strategy using iProof high-fidelity DNA polymerase (Bio-Rad Laboratories). The influenza virus hemagglutinin (HA) tag was inserted between S986 and L987 of hNPC1L1.
The pcDNA3.1 constructs encoding rNPC1L1, hNPC1L1-EGFP, hNPC1L1L1072T/L1168I-EGFP, HA-hNPC1L1-EGFP, or HA-hNPC1L1L1072T/L1168I-EGFP were transfected into HEK293 cells using FuGENE HD (Promega). Cells were selected by culturing in the presence of 1 mg/ml G418 sulfate (Wako Pure Chemical Industries). For FACS analysis, HEK293 cells stably expressing HA-hNPC1L1L1072T/L1168I-EGFP were cloned by limiting dilution.
3
FBN1-ZFNs Target Region Amplification
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The target region of FBN1-ZFNs was amplified via direct PCR from the cell clones using MightyAmp DNA polymerase (Takara Bio, Shiga, Japan) and the corresponding primers 5′-GACATAGGTGAAGACTTCGTAGG and 5′-TCACTCTCAAGACTCCAGTTTGG. Nested PCR was performed using PrimeSTAR HS DNA polymerase (Takara Bio) and the appropriate primers 5′-AACTGAGAGTGACTTCCATGGAC and 5′-GCAACCGCTCATTTTCCTCTATG. The nested PCR fragment from each clone was analysed using the sequencing primer 5′-TAACTTGTGCTCCAGCTCAGGTG, the BigDye Terminator Cycle Sequencing Kit and an ABI PRISM 3130xl Genetic Analyzer (Life Technologies). The nested PCR fragments that contained the mutation were subcloned into the sequencing vector pCR4Blunt-TOPO (Life Technologies).
For analysis of the mutation in each piglet, genomic DNA was extracted from tail biopsies of the piglets using a DNeasy Tissue and Blood Kit (QIAGEN, Hilden, Germany), and PCR genotyping and DNA sequencing were performed as described above.
For analysis of the mutation in each piglet, genomic DNA was extracted from tail biopsies of the piglets using a DNeasy Tissue and Blood Kit (QIAGEN, Hilden, Germany), and PCR genotyping and DNA sequencing were performed as described above.
4
Yeast Genomic and Plasmid DNA Isolation
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For PCR, genomic or plasmid DNA from yeast was isolated by resuspending cells in 250 µl P1 (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/ml RNase A), adding 0.5-mm glass beads to displace an additional ∼250 µl of volume and vortexing for 3 min prior to addition of P2 (200 mM NaOH, 1% SDS) and N3 (4.2 M Guanidinium-HCl, 0.9 M potassium acetate, pH 4.8). Lysates were clarified by centrifugation and applied to Zyppy plasmid DNA miniprep columns (Zymo Research); then DNA was washed and eluted according to the manufacturer’s instructions. Plasmids were transformed to competent bacterial cells using the Mix & Go E. coli Transformation Kit (Zymo Research) or supercompetent XL1-Blue cells according to the manufacturer’s instructions. PCR was performed with various high-fidelity enzymes, typically Q5 (New England Biolabs), according to the manufacturer’s instructions. Primers and nucleotides were eliminated prior to dideoxy sequencing by treatment of the PCR reaction with ExoI and FastAP (Fermentas), according to the manufacturer’s instructions. For those PCR products that were TOPO® cloned, pCR4Blunt-TOPO® (Life Technologies) was used according to the manufacturer’s instructions. PFGE and array-based comparative genomic hybridization (array-CGH), along with yeast DNA preparation for those techniques, were performed as described previously (Argueso et al. 2008 (link)).
5
Cloning and Expression of AlHV-1 gB
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The gene encoding gB (ORF8 5 ) from AlHV-1 was expressed in recombinant form as follows: ORF8 was amplified from AlHV-1 strain C500 DNA by PCR using primers ORF8FwdAsc (5'-GGC GCG CCG GCG CAC ACA GGT AGC ACA GTC T-3'), ORF8RevFse (5'-GGC CGG CCC GAC ACC AGT GCT CTC AAA AGA GT-3') and KOD hot-start DNA polymerase (Merck, Nottingham, UK). The 2.6 kbp fragment was inserted into pCR4-blunt-TOPO (Life Technologies) and the DNA sequence of the resulting clones was determined using flanking and internal primers. A verified ORF8 fragment was excised using restriction enzymes AscI and NgoMIV and inserted into the mammalian expression vector pVR-MCS-HA, derived from pVR1255 (a kind gift from Vical Inc. 19 ).
To generate pVR-MCS-HA, a synthetic 218 bp double-stranded cassette encoding a ribosome binding site with start codon, a multiple cloning site, a TEV protease cleavage site and a C-terminal triple-HA (haemagglutinin) epitope tag (TOP Gene Technologies, Montreal Canada; accession number FN553440) was inserted between the NotI and BamHI sites of pVR1255. The correct structure of the resulting AlHV-1 gB expression vector (pVR-ORF8-HA) was confirmed by restriction enzyme analysis.
To generate pVR-MCS-HA, a synthetic 218 bp double-stranded cassette encoding a ribosome binding site with start codon, a multiple cloning site, a TEV protease cleavage site and a C-terminal triple-HA (haemagglutinin) epitope tag (TOP Gene Technologies, Montreal Canada; accession number FN553440) was inserted between the NotI and BamHI sites of pVR1255. The correct structure of the resulting AlHV-1 gB expression vector (pVR-ORF8-HA) was confirmed by restriction enzyme analysis.
6
Quantification of Murine Thymic Regulatory T Cells
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To obtain splenic CD4 single positive (SP) and CD8SP T cells, red blood cells were lysed using a RED BLOOD CELL LYSING BUFFER (Sigma, St. Louis, MO) followed by the depletion of CD19, Mac1 and γδT cells with magnetic beads. CD4SP cells and CD8SP cells were sorted by a FACS AriaⅢ (Becton Deckinson). Sorted cells were lysed in ProteinaseK (Sigma, St. Louis, MO) to prepare genomic DNA. An mTREC were quantified as previously described [74 ]. An mTREC standard for the real-time PCR was generated by PCR cloning of a 586-base-pair fragment of mTREC DNA from C57BL/6 mouse thymus genomic DNA into pCR4Blunt-TOPO (Invitrogen, Carlsbad, CA). An mTREC of splenic CD4SP and CD8SP cells were amplified and quantified by ABI StepOnePlus using Power SYBR Green PCR Master Mix (Applied Biosystems). Primers used to detect mTREC were as follows: Forward primer: 5’ -TCATTGCCTTTGAACCAAGC- 3’, Reverse primer: 5’–CACAGCAGCTGTGGGTTTATG- 3’
7
Plasmid Purification and Sequencing from S. aureus
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Purification of plasmids from S. aureus strains positive for ses and set was carried out as described
previously [16 (link), 18 (link)]. The TOPO shotgun cloning kit (Invitrogen, Carlsbad, CA, USA),
pCR4®Blunt-TOPO® (Invitrogen) and One shot® TOPO10 electrocompTME. coli (Invitrogen) were used
for preparation of the shotgun library. White colonies were cultured in LB broth (Sigma Aldrich, St. Louis, MO, USA) containing 100 µg/ml ampicillin (Wako Pure
Chemical Industries, Osaka, Japan) and cultured at 37°C under shaking conditions overnight. The cultured cells were subjected to plasmid extraction using the
QIAprep8 (QIAGEN, Hilden, Germany) system. Nucleotide sequences of the shotgun library were obtained using an automatic DNA sequencer ABI3100Avant (Applied
Biosystems, Foster City, CA, USA) and assembled using AGCT software, ver. 4.0 (Genetyx, Tokyo, Japan). Gaps of contigs were closed by primer walking with
primers designed at contig ends.
previously [16 (link), 18 (link)]. The TOPO shotgun cloning kit (Invitrogen, Carlsbad, CA, USA),
pCR4®Blunt-TOPO® (Invitrogen) and One shot® TOPO10 electrocompTME. coli (Invitrogen) were used
for preparation of the shotgun library. White colonies were cultured in LB broth (Sigma Aldrich, St. Louis, MO, USA) containing 100 µg/ml ampicillin (Wako Pure
Chemical Industries, Osaka, Japan) and cultured at 37°C under shaking conditions overnight. The cultured cells were subjected to plasmid extraction using the
QIAprep8 (QIAGEN, Hilden, Germany) system. Nucleotide sequences of the shotgun library were obtained using an automatic DNA sequencer ABI3100Avant (Applied
Biosystems, Foster City, CA, USA) and assembled using AGCT software, ver. 4.0 (Genetyx, Tokyo, Japan). Gaps of contigs were closed by primer walking with
primers designed at contig ends.
8
Extrachromosomal Expression of PTEN-Halo
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Plasmids for the extrachromosomal expression of PTENi-Halo were constructed based on the same vector as previously reported [26] (link). Mutations for basic amino acids substitutions in the cα2 helix were introduced into the pten gene cloned into pCR4BluntTOPO (Invitrogen) according to the manufacturer's manual (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent Technologies). The primers used are shown in Table 1 . The resultant nucleotide sequences were confirmed by sequencing. The mutant genes were cloned into the BglII and SpeI sites of pHK12neo to generate in-frame fusion with the gene encoding HaloTag between the promoter and terminator sequences. 5 µg of the expression plasmids were used for the transformation. Transformation of the plasmids for the expression of PTEN-Halo or PTENi-Halo was performed as described previously [26] (link). The transformed cells were selected under 10 µg/ml of G418 for 1 week.
9
Deletion Cassette Construction and Transformation
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The upstream (LB-BIP1; 1.2 kb) and downstream (RB-BIP1; 1.36 kb) regions flanking BIP1 were obtained by PCR amplification using P1.2 genomic DNA as a template, Pfu turbo polymerase (Stratagene, La Jolla, CA) and the couples of primers KO1/KO2-SfiIa and KO3-SfiIb/KO4, respectively (S1 Fig S5 Table
10
Cloning and Expression of env Gene
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PCR products were cloned directly into the pCR4Blunt-TOPO (Invitrogen, Carlsbad, CA, USA) or pUC118 vector (Mighty Cloning Kit; Takara). The intact env gene was inserted into the pFUΔss expression vector (52 (link)). Sequencing was performed using a contracted service (Fasmac Corporation, Atsugi, Japan).
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