Journal: PLoS ONE

Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

doi: 10.1371/journal.pone.0018900

Figure Lengend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

Techniques: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

Journal: PLoS ONE

Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

doi: 10.1371/journal.pone.0018900

Figure Lengend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

Techniques: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: SMAD2 Inactivation Inhibits CLDN6 Methylation to Suppress Migration and Invasion of Breast Cancer Cells

doi: 10.3390/ijms18091863

Figure Lengend Snippet: DNA methyltransferase 1 (DNMT1)-mediated upregulation of CLDN6 expression by SB431542. ( A , B ) Real-time polymerase chain reaction (RT-PCR) and immunoblot analysis of DNMT1 and CLDN6, and densitometric analysis of relative gene expression levels after normalization to loading controls GAPDH and β-actin are presented. ( C ) DNMT1 activity assays. ( D ) Methylation-specific PCR (MSP) analysis of CpG island of CLDN6 promoter using bisulfite-treated genomic DNA isolated from MCF-7 and SKBR-3 cells. ( E ) CpG island methylation within the CLDN6 promoter region was measured by bisulfite sequencing in SKBR-3 cells. “Me” stands for methylated, and “U” stands for unmethylated. ( F ) Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay to detect the binding of DNMT1 to the promoter of CLDN6 (** p

Article Snippet: Antibodies included anti-DNMT1 (1:500, Cell Signaling Technology), anti-IgG (Millipore), and anti-DNA polymerase II (Millipore); anti-IgG was the negative control and anti-DNA polymerase II was the positive control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Methylation, Polymerase Chain Reaction, Isolation, Methylation Sequencing, Chromatin Immunoprecipitation, Binding Assay

Journal: PLoS Genetics

Article Title: The Broad-Spectrum Antiviral Protein ZAP Restricts Human Retrotransposition

doi: 10.1371/journal.pgen.1005252

Figure Lengend Snippet: ZAP inhibits exogenous L1 RNP levels in cells. (A) Expression of HA-ZAP-L, HA-ZAP-S, and V5-TEV-MOV10 reduces the amount of ORF1 protein (top IP panel) and ORF2p RT activity (bottom IP panel) in α-FLAG antibody purified pc-L1-1FH immunoprecipitates. RT activity was determined by the LEAP assay [ 81 ]. Below: Coomasie-stained gel of cell lysates prior to IP showed no obvious loss of global protein levels in the presence of ZAP. (B) Analysis of 293T cell lysates showing that ORF1 protein from pc-L1-1FH is reduced in the presence of ZAP and MOV10, but not empty vector or an unrelated protein, C22ORF28 (top panel). Panels below show by Western blotting that neither ZAP nor MOV10 alter expression of endogenous HSP90 or GFP protein expressed from cotransfected CEP-EGFP. Lysates were prepared from two pooled wells of a six-well plate. (C) Expression of ZAP causes loss of L1 RNA. Top panels: RT-PCR of L1 RNA expressed from 99-PUR-JM111-EGFP, which expresses L1-RP with an ORF1 mutation that prevents genomic insertion. PCR primers spanned the intron of the EGFP reporter cassette to distinguish spliced RNA products from contaminating plasmid DNA (generating a 105 nt band vs a 1 kb band). PCR reactions are shown in the presence or absence of RT. Bottom panels: Levels of endogenous HSPA6 RNA were the same in the presence or absence of ZAP.

Article Snippet: Subsequent PCR used GoTaq DNA polymerase (Promega).

Techniques: Expressing, Activity Assay, Purification, Staining, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

doi: 10.1038/srep05052

Figure Lengend Snippet: Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

Article Snippet: Better performance of AccuPrime Pfx DNA polymerase compared to other DNA polymerases on repetitive DNA templates is also likely due to its mixture of other proprietary thermostable accessory proteins in this polymerase mix ( http://tools.lifetechnologies.com/content/sfs/manuals/accuprimepfx_man.pdf ) .

Techniques: Binding Assay, Amplification, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

doi: 10.1038/s41598-018-19930-w

Figure Lengend Snippet: Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

Article Snippet: Primers were purchased from GCC biotech (India) while PCR master mix and hot start DNA polymerase was procured from Sigma, USA.

Techniques: Irradiation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence, Inhibition, MTT Assay

Journal: BMC Biotechnology

Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

doi: 10.1186/1472-6750-11-92

Figure Lengend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

Article Snippet: The PCR reaction components were: 50 μl total volume, 0.5 μl Phusion DNA polymerase (New England Biolabs, Ipswich, MA), or 0.8 μl Pfu Turbo, or PfuUltra DNA polymerase (Agilent Technologies, Inc, Santa Clara, CA), 5 μl 10× buffer; 5 μl of 2.5 mM dNTPs; 10 ng of plasmid DNA template; 5 pmol of each primer.

Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: CXCL8 histone H3 acetylation is dysfunctional in airway smooth muscle in asthma: regulation by BET

doi: 10.1152/ajplung.00021.2015

Figure Lengend Snippet: CXCL8 hypersecretion is not associated with differences in DNA methylation. Genomic DNA was isolated from ASM cells from 3 asthmatic and 3 nonasthmatic individuals and bisulfite converted. Regions of DNA containing CpG sites were PCR amplified and pyrosequenced. A : schematic showing the position of the 8 CXCL8 CpG sites, the PCR primers (gray arrows), and sequencing primers (black arrows). B : percent methylation of the individual CpG sites in ASM cells from both asthmatic and nonasthmatic individuals.

Article Snippet: PCRs were performed with the HotStarTaq DNA polymerase PCR kit (Qiagen) under the following conditions: 95°C 5 min; 45 cycles of 94°C 30 s; 56°C 30 s; 72°C 30 s; finally 72°C 10 min, except the PCR primers for CpGs 7 and 8, which required an annealing temperature of 52.6°C.

Techniques: DNA Methylation Assay, Isolation, Polymerase Chain Reaction, Amplification, Sequencing, Methylation

Journal: Balkan Journal of Medical Genetics : BJMG

Article Title: UGT1A1 (TA)n Promoter Genotype: Diagnostic and Population Pharmacogenetic Marker in Serbia

doi: 10.2478/bjmg-2018-0012

Figure Lengend Snippet: (a) UGT1A1 (TA) n promoter PCR products on 15.0% acrylamide gel electrophoresis stained with Ag-nitrate. Wells 1 and 4: 6/6 TA repeats (71 bp); well 2: 7/7 TA repeats (73 bp); well 5: 6/7 TA repeats (71 and 73 bp); well 3: 5 bp DNA ladder. (b) Electophoretograms obtained with fragment length analysis of UGT1A1 (TA) n promoter repeats. The upper peak is 7/7 TA repeats (102 bp); the lower peak is 6/6 TA repeats (100 bp). (c) Electophoretogram obtained with sequencing analysis of promoter of UGT1A1 gene with 6/6 TA repeats. (d) Electophoretogram obtained with sequencing analysis of promoter of UGT1A1 gene with 7/7 TA repeats.

Article Snippet: The amplification reaction was performed in a total volume of 30 μL, and the reaction mix contained 10 pmol of each primer, 50-100 ng of genomic DNA, 0.5 mM of each dNTP (Fermentas), 1 × PCR reaction buffer, 1.4 mM MgCl2 , 1 U DNA polymerase (KAPA Biosystems, Wilmington, MA, USA).

Techniques: Polymerase Chain Reaction, Acrylamide Gel Assay, Electrophoresis, Staining, Sequencing

Journal: Iranian Journal of Basic Medical Sciences

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

doi:

Figure Lengend Snippet: Antigenic region of the VacA gene from Helicobacter pylori amplified by PCR. Lane 1: DNA marker, Lane 2: PCR product

Article Snippet: PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Techniques: Amplification, Polymerase Chain Reaction, Marker

Journal: Frontiers in Genetics

Article Title: Simultaneous Methylation-Level Assessment of Hundreds of CpG Sites by Targeted Bisulfite PCR Sequencing (TBPseq)

doi: 10.3389/fgene.2017.00097

Figure Lengend Snippet: Evaluation of the targeted bisulfite PCR sequencing (TBPseq) method. (A) Illustration of TBPseq method. Colored horizontal arrows denote primers for target amplification. Amplicons have methylated (5′-CG-3′) or unmethylated (5′-TG-3′) sequences for target CpGs, the ratio of which is used for calculating methylation frequencies of the target CpGs. A total of 113 primer pairs were split into six groups of about 20 pairs for separate multiplexed PCR. (B) Strategy for evaluating the TBPseq method. The whole genomic DNA of 293T cells was amplified using ϕ29 DNA polymerase. A half of the resulting newly synthesized and unmodified (demethylated) DNA was re-methylated in vitro using CpG dinucleotide-specific Sss I methylase. The remethylated and demethylated DNA fractions were equally treated with bisulfite and used as template in multiplex PCR with 20–25 primer pairs per reaction. Multiplex PCR was additionally performed with an equal mixture (1:1 mixed DNA) of the remethylated and demethylated DNA templates. The three different groups of amplicons were modified and differentially barcoded for Illumina sequencing. (C) Methylation levels of target CpG sites. Each CpG site has three different methylation levels that were obtained from demethylated DNA (ϕ29 only, blue), remethylated DNA (ϕ29 + SssI, red), and an equal mixture of them (purple). In the right panel, the mean methylation levels of target CPGs in the three DNA groups are shown (error bars, standard deviation). Individual CpG sites are indicated by their genomic position as “chromosome (chr) number:coordinate” using GRCh37/hg19 as reference genome.

Article Snippet: 5′-end phosphorylation was performed with 1 μg of pooled amplicons using T4 polynucleotide kinase (NEB) at 37°C for 30 min. 5′-end phosphorylated amplicons then were ligated with 15 μM of home-made Illumina adaptors by incubating them at RT for 1 h. Finally, adaptor ligated amplicons were amplified using index and universal primers by DNA polymerase (SolGent) for indexing in the following conditions: 15 min of enzyme activation at 98°C followed by 25 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 30 s ( ).

Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Methylation, Synthesized, In Vitro, Multiplex Assay, Modification, Standard Deviation

Journal: Journal of Bacteriology

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA

doi: 10.1128/JB.185.17.5210-5219.2003

Figure Lengend Snippet: Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.

Article Snippet: An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 .

Techniques: Clone Assay, Polymerase Chain Reaction, Generated, Southern Blot, Staining, Agarose Gel Electrophoresis, Sequencing