Structured Review

Thermo Fisher t4 pnk
Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Strains were transformed with plasmid pMK184 (). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a <t>T4</t> PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a
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Images

1) Product Images from "DNA-guided DNA interference by a prokaryotic Argonaute"

Article Title: DNA-guided DNA interference by a prokaryotic Argonaute

Journal:

doi: 10.1038/nature12971

Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Strains were transformed with plasmid pMK184 (). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a
Figure Legend Snippet: Analyses of TtAgo in T. thermophilus and E.coli a , TtAgo decreases plasmid transformation efficiency of T. thermophilus . Transformation efficiency of different ago mutant strains relative to the transformation efficiency of wild-type strain HB27. HB27 EC is an HB27 mutant selected for high competence, and HB27Δ ago is an ago ). Strains were transformed with plasmid pMK184 (). Transformations were performed in biological duplicates for each strain. Error bars indicate standard deviations. b , Effect on TtAgo expression on plasmid content in E. coli KRX. TtAgo and TtAgoDM were expressed in E. coli KRX from plasmid pWUR702 and pWUR703. Plasmids were purified from biological triplicate cultures in which expression was induced (+) or not induced (−). Compared with TtAgoDM expression, TtAgo expression in E. coli KRX does not lead to reduced plasmid content. Changes in plasmid yield between induced and not induced cultures probably originate from protein expression energy costs. Error bars indicate standard deviations. c , 10–150-nucleotide (nt) RNA with 5′-OH group co-purifies with TtAgo. 15% denaturing polyacrylamide gels with nucleic acids co-purified with TtAgo and TtAgoDM. Nucleic acids are phosphorylated in a T4 PNK forward reaction (5′-OH groups, and to a lesser extend 5′-P groups, are labelled) using [γ- 32 P] ATP, and resolved on 15% denaturing polyacrylamide gels. Nucleic acids were not treated (lane 1, 5), RNaseA treated (lanes 2, 6), DNaseI treated (lane 3, 7) or Nuclease P1 treated (lane 4, 8). Fig. 1a

Techniques Used: Plasmid Preparation, Transformation Assay, Mutagenesis, Expressing, Purification

2) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039251

12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4  and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.
Figure Legend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

Techniques Used: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.
Figure Legend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B  = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

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Centrifugation:

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Amplification:

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Whole Genome Amplification:

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Purification:

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Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
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Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
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Autoradiography:

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Construct:

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
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Real-time Polymerase Chain Reaction:

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Multiplex Assay:

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: The PCR consisted of 25 cycles of amplification with priming temperature and elongation time optimized for each amplicon multiplex. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase.

Incubation:

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min.

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: Fifty nanograms of genomic DNA and 9 μl of a sample buffer containing random hexamer primers were heat denatured and cooled, allowing random priming of the hexamers, then 9 μl of reaction buffer and 1 μl of Phi29 DNA polymerase were added and incubated overnight at 30°C for linear DNA synthesis. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase.

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: After incubation at 22°C and 95 to 100% humidity for 48 h, ticks were either dissected to test for protein or transcript knockdown or fed 8 μl of a rickettsial solution containing 30,000 rickettsiae/μl as described above and incubated at 22°C and 95 to 100% humidity for 24 h. In short, transcript and protein levels for DvKPI were assessed 48 h after delivery of the siRNA, after which ticks were fed the rickettsial suspension. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis.

Article Title: Co-operative binding assay for the characterization of mGlu4 allosteric modulators
Article Snippet: The SDS PAGE was done using 20 μg of protein sample and the proteins were transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). .. After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with antibodies against mGlu4 (1:2000) at 4 °C for 12 h. Membranes were washed three times for 10 min and incubated with a secondary antibody (1:5000 dilution) ECL Anti-rabbit IgG (horseradish peroxidase linked whole antibody from donkey NA934V (GE Healthcare UK)) for 1 h. Blots were washed with TBST three times and developed (#34080 Thermo Supersignal West Pico Chemiluminescent substrate) on HyBlot CL Autoradiography Film (Cat No.: E3012 Denvill Scientific Inc.). .. CHO cells expressing mGlu4 were used for all binding assay protocols.

Activity Assay:

Article Title: Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members
Article Snippet: We observed that both highly concentrated stocks as well as working solutions of LNAzymes partly lose their activity after storage at −20°C for several months. .. The LNAzyme digestion buffer contained 100 mM NaCl, 50 mM RNase-free Tris (pH 8.0) (Ambion, Austin, TX), 10 mM LiCl, 1 μM LNAzyme, and 5 μl of an in vitro transcription reaction mixture containing the transcribed RNA (see above).

Infection:

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: Measurement of DvKPI levels and abundance was not performed at the same time post-rickettsial infection because these studies were focused on the effect of DvKPI suppression as it relates to rickettsial entry. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis.

Expressing:

Article Title: Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions
Article Snippet: Paragraph title: Quantitative PCR for the expression of pro- and anti-apoptotic macrophage genes ... Then 1 μg of purified RNA was mixed with 10 μl of a solution consisting of a basic buffer (100 mM Tris-HCl, pH 8.3, containing 500 mM KCl and 15 mM MgCl2 Invitrogen, USA), dNTP (10mM; Fermentas, USA), random primers (Invitrogen, USA), OligoDT primers (Invitrogen, USA), RNaseOUT recombinant ribonuclease inhibitor (40 U μl−1 ; Invitrogen, USA), M-MLV reverse transcriptase (100 U μl−1 ).

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: All purification procedures were carried out at 4°C. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM). .. Fractions containing denatured RecD where loaded onto Superdex 200 equilibrated with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 6 M Urea.

BIA-KA:

Article Title: RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model
Article Snippet: Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized. .. Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized.

Western Blot:

Article Title: IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System
Article Snippet: Paragraph title: Western blotting ... Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL).

Article Title: The functional significance of microRNA-145 in prostate cancer
Article Snippet: Paragraph title: Western analysis ... Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing 1 × protease inhibitor cocktail (Roche, Basel, Switzerland).

Article Title: Transfection of microRNA Mimics Should Be Used with Caution
Article Snippet: Data were analyzed using the ImageQuant software. .. HeLa cells were lysed in 1% NP-40 lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, 1 mM DTT and proteinase inhibitors (halt inhibitor, Thermo #78442), and subjected to separation on 8 or 10% SDS-PAGE gels and Western blot (10 μg whole cell lysate per lane). .. Antibodies used for Western blot were anti-Bim (Cell Signaling, 2933), anti-Pten (Cell Signaling, 9559), anti-Phlpp2 (Bethyl, A300-661A-1), and anti-Ship1 (Cell Signaling, 2728).

Article Title: RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model
Article Snippet: Paragraph title: Western Blotting ... Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized.

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis.

Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
Article Snippet: Paragraph title: Western blotting ... AA6-treated/untreated 4T1-injected mice tumorigenic tissue (10–25 mg) and AA6-treated/untreated 4T1 cells samples were lysed in Laemmli buffer (Tris HCl 100 mM pH 6.8, SDS 4%, glycerol 20%, DTT 25 mM, NuPAGE LDS Sample Buffer 1x - Invitrogen).

Article Title: Co-operative binding assay for the characterization of mGlu4 allosteric modulators
Article Snippet: Paragraph title: 2.2.3 Western blot ... After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with antibodies against mGlu4 (1:2000) at 4 °C for 12 h. Membranes were washed three times for 10 min and incubated with a secondary antibody (1:5000 dilution) ECL Anti-rabbit IgG (horseradish peroxidase linked whole antibody from donkey NA934V (GE Healthcare UK)) for 1 h. Blots were washed with TBST three times and developed (#34080 Thermo Supersignal West Pico Chemiluminescent substrate) on HyBlot CL Autoradiography Film (Cat No.: E3012 Denvill Scientific Inc.).

Article Title: The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus
Article Snippet: Nuclei were lysed in 100μl of Lysis Buffer X (LBX) (50mM Tris pH 8.0, 2.5mM EDTA pH 8.0 (Invitrogen), 0.25% SDS (Euromedex)) in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche). .. The lysates were sonicated for 20 minutes at 4°C in a Sonorex Digitec (model DT100) ultrasonic bath (Bandelin).

Transformation Assay:

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The PCR cycling conditions were as follows: 10 min at 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C, and a final elongation for 5 min at 72 °C.

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: Gene insertion was verified via sequencing, then heat-shock transformed into BL21 bacteria. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Derivative Assay:

Article Title: The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus
Article Snippet: 2 million MDDCs at day 4 or day 5 post-differentiation or mouse bone marrow derived DCs at day 10 post-differentiation were collected, washed with 1ml of PBS, and processed according to two different fractionation protocols. .. Nuclei were lysed in 100μl of Lysis Buffer X (LBX) (50mM Tris pH 8.0, 2.5mM EDTA pH 8.0 (Invitrogen), 0.25% SDS (Euromedex)) in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche).

Electron Microscopy:

Article Title: Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones
Article Snippet: The LoopAmp RNA amplification kit contains 2× Reaction Mix (RM) (40 mM Tris-HCl, pH 8.8, 20 mM KCl, 16 mM MgSO4 , 20 mM (NH4 )2 SO4 , 0.2% Tween 20, 1.6 M betaine, and dNTPs 2.8 mM each), Enzyme Mix (EM) (mixture of Bst DNA polymerase and AMV reverse transcriptase), and distilled water (DW). .. Tris-HCl buffer stock solution (1 M, pH 8.0) was purchased from Affymetrix (Santa Clara, CA, USA).

Ligation:

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The PCR cycling conditions were as follows: 10 min at 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C, and a final elongation for 5 min at 72 °C.

Protease Inhibitor:

Article Title: miR-128 inhibits telomerase activity by targeting TERT mRNA
Article Snippet: Briefly, 10 mm plates of 80% confluent cultured cells were washed with buffer A [20 mM Tris-HCl pH 8.0, 280 mM KCl, 10 mM EDTA, 1% NP-40, 0.2% Deoxycholate, 2X Halt protease inhibitor cocktail (Pierce), 200 U/ml RNaseout (ThermoFisher Scientific) and 1 mM DTT]. .. Protein concentration was adjusted across samples with buffer B [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 0.1% deoxycholate, 100 U/ml Rnaseout (ThermoFisher Scientific), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)]. .. Lysates were centrifuged at 16,000xg for 15 min at 4° C and supernatants were incubated with 10–20 µg of 4F9 antibody conjugated to epoxy magnetic beads (M-270 Dynabeads, ThermoFisher) for 2 hours at 4° C with gentle rotation.

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: 20×106 cells were trypsinized and crosslinked with 1% paraformaldehyde (w/v) (ThermoFisher Scientific) at room temperature for 5 min, followed by 125 mM glycine (Sigma) for 5 min to quench the crosslinking and washed (cold 1× PBS, 1 mM PMSF). .. Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Nuclei pellets were incubated in nuclear lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA with 1× Halt protease inhibitor cocktail) for 30 min, further lysed with a syringe, and sonicated with Covaris S2 to obtain fragments between 400 and 1000 base pairs.

Article Title: IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System
Article Snippet: Cells were harvested by the treatment of trypsin-EDTA. .. Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL). .. Whole cells and cell debris were pelleted by low speed centrifugation and cleared supernatants were transferred to a new tube.

Article Title: The functional significance of microRNA-145 in prostate cancer
Article Snippet: Stained cells were immediately analysed by flow cytometry (Cell Lab Quanta SC; Beckman Coulter). .. Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing 1 × protease inhibitor cocktail (Roche, Basel, Switzerland). .. Protein estimations were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions.

Article Title: RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model
Article Snippet: The processed gene expression data have been submitted to the NCBI Gene Expression Omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE56227. .. Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized. .. Ear homogenates were centrifuged at 12000 × g for 10 minutes.

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Cultures were grown to late log phase, harvested by centrifugation, and weighed. .. Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). .. The soluble fraction was collected as S20 cell extract, with protein quantified by Qubit assay (Life Technologies).

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: Tick midguts were dissected, placed in RLT extraction buffer (Qiagen), and stored at −80°C until used for RNA isolation. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis. .. Quantitative RT-PCR was used to determine transcript and rickettsial abundance as described above.

Article Title: The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus
Article Snippet: The recovered supernatant represented the cytosolic fraction and were stored on ice. .. Nuclei were lysed in 100μl of Lysis Buffer X (LBX) (50mM Tris pH 8.0, 2.5mM EDTA pH 8.0 (Invitrogen), 0.25% SDS (Euromedex)) in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche). .. The lysates were sonicated for 20 minutes at 4°C in a Sonorex Digitec (model DT100) ultrasonic bath (Bandelin).

Cell Culture:

Article Title: miR-128 inhibits telomerase activity by targeting TERT mRNA
Article Snippet: Briefly, 10 mm plates of 80% confluent cultured cells were washed with buffer A [20 mM Tris-HCl pH 8.0, 280 mM KCl, 10 mM EDTA, 1% NP-40, 0.2% Deoxycholate, 2X Halt protease inhibitor cocktail (Pierce), 200 U/ml RNaseout (ThermoFisher Scientific) and 1 mM DTT]. .. Protein concentration was adjusted across samples with buffer B [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 0.1% deoxycholate, 100 U/ml Rnaseout (ThermoFisher Scientific), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)].

Generated:

Article Title: Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions
Article Snippet: Then 1 μg of purified RNA was mixed with 10 μl of a solution consisting of a basic buffer (100 mM Tris-HCl, pH 8.3, containing 500 mM KCl and 15 mM MgCl2 Invitrogen, USA), dNTP (10mM; Fermentas, USA), random primers (Invitrogen, USA), OligoDT primers (Invitrogen, USA), RNaseOUT recombinant ribonuclease inhibitor (40 U μl−1 ; Invitrogen, USA), M-MLV reverse transcriptase (100 U μl−1 ). .. The reactions included master mix Syber Green (2×) (Applied Biosystems, USA) and 1 μl cDNA (1 μg) template and were run in triplicate on a real-time PCR system (StepOne; Applied Biosystems, USA).

other:

Article Title: Assaying RNA structure with LASER-Seq
Article Snippet: For E. coli experiments, pellets were resuspended in water and split for reverse transcription with either TGIRTIII (Ingex) or SSII (Invitrogen).

DNA Sequencing:

Article Title: Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme
Article Snippet: Paragraph title: PCR amplification and DNA sequencing ... All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample.

Protein Concentration:

Article Title: miR-128 inhibits telomerase activity by targeting TERT mRNA
Article Snippet: Briefly, 10 mm plates of 80% confluent cultured cells were washed with buffer A [20 mM Tris-HCl pH 8.0, 280 mM KCl, 10 mM EDTA, 1% NP-40, 0.2% Deoxycholate, 2X Halt protease inhibitor cocktail (Pierce), 200 U/ml RNaseout (ThermoFisher Scientific) and 1 mM DTT]. .. Protein concentration was adjusted across samples with buffer B [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 0.1% deoxycholate, 100 U/ml Rnaseout (ThermoFisher Scientific), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)]. .. Lysates were centrifuged at 16,000xg for 15 min at 4° C and supernatants were incubated with 10–20 µg of 4F9 antibody conjugated to epoxy magnetic beads (M-270 Dynabeads, ThermoFisher) for 2 hours at 4° C with gentle rotation.

Article Title: IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System
Article Snippet: Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL). .. Whole cells and cell debris were pelleted by low speed centrifugation and cleared supernatants were transferred to a new tube.

Article Title: A proteomic landscape of diffuse-type gastric cancer
Article Snippet: Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%). .. Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%).

Article Title: RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model
Article Snippet: Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized. .. Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized.

Sequencing:

Article Title: Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme
Article Snippet: All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample. .. PCR products were then purified with both Exonuclease I (Biolabs, England) and Shrimp Alkaline Phosphatase (Biolabs, England) as per manufacturer’s instructions.

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The plasmid DNAs were purified after color selection.

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: Gene insertion was verified via sequencing, then heat-shock transformed into BL21 bacteria. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Sonication:

Article Title: A proteomic landscape of diffuse-type gastric cancer
Article Snippet: Specimen in which EBER nuclear expression was observed in > 20% of the tumor cells were considered EBER positive. .. Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%). .. The lysate was centrifuged at 14,000×g for 10 min and the supernatant was collected as whole tissue extract.

Recombinant:

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). .. Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells).

Article Title: Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions
Article Snippet: To evaluate the expressions of pro- and anti-apo ptotic macrophage genes, total RNA was extracted from 2 × 105 cells ml−1 using TRIzol (Invitrogen, USA), following the manufacturer's protocol (RNA integrity was determined as an OD260/280 absorption ratio between 1.8 and 2.1). .. Then 1 μg of purified RNA was mixed with 10 μl of a solution consisting of a basic buffer (100 mM Tris-HCl, pH 8.3, containing 500 mM KCl and 15 mM MgCl2 Invitrogen, USA), dNTP (10mM; Fermentas, USA), random primers (Invitrogen, USA), OligoDT primers (Invitrogen, USA), RNaseOUT recombinant ribonuclease inhibitor (40 U μl−1 ; Invitrogen, USA), M-MLV reverse transcriptase (100 U μl−1 ). .. The reactions were incubated at 37°C for 50 min and were denatured at 70°C for 15 min. For real-time quantitative RT-PCR, the following primers set were designed: murine Bax gene (GenBank Accession No. NM007527), forward: 5′-GGC CTT TTT GCT ACA GGG TTT CAT-3′ and reward: 5′-TGC TGT CCA GTT CAT CTC CAA TTC-3′; for murine Bcl-2 gene, forward: 5′-GAC TGA GTA CCT GAA CCG GCA TCT-3′ and reward: 3′-AAG CCC AGA CTC ATT CAA CCA GAC-3′ (GenBank Accession No. NM009741), and for murine IGF-I, forward, 5′-TAC TTC AAC AAG CCC ACA GG-3′ and reward, 5′-AGT CTT GGG CAT GTC AGT GT-3′ (GenBank Accession No. NM010512). β-Actin (GenBank Accession No. NM00739) was used as a constitutively expressed control gene for normalization (primers: forward, 5′-GCC TTC CTT CTT GGG TAT GGA ATC-3′ and reward, 5′-ACG GAT GTC AAC GTC ACA CTT CAT-3′).

Imaging:

Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
Article Snippet: AA6-treated/untreated 4T1-injected mice tumorigenic tissue (10–25 mg) and AA6-treated/untreated 4T1 cells samples were lysed in Laemmli buffer (Tris HCl 100 mM pH 6.8, SDS 4%, glycerol 20%, DTT 25 mM, NuPAGE LDS Sample Buffer 1x - Invitrogen). .. AA6-treated/untreated 4T1-injected mice tumorigenic tissue (10–25 mg) and AA6-treated/untreated 4T1 cells samples were lysed in Laemmli buffer (Tris HCl 100 mM pH 6.8, SDS 4%, glycerol 20%, DTT 25 mM, NuPAGE LDS Sample Buffer 1x - Invitrogen).

DNA Synthesis:

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: Fifty nanograms of genomic DNA and 9 μl of a sample buffer containing random hexamer primers were heat denatured and cooled, allowing random priming of the hexamers, then 9 μl of reaction buffer and 1 μl of Phi29 DNA polymerase were added and incubated overnight at 30°C for linear DNA synthesis. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase.

Fractionation:

Article Title: The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus
Article Snippet: Paragraph title: Nuclear-Cytoplasmic fractionation ... Nuclei were lysed in 100μl of Lysis Buffer X (LBX) (50mM Tris pH 8.0, 2.5mM EDTA pH 8.0 (Invitrogen), 0.25% SDS (Euromedex)) in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche).

Mutagenesis:

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: The PCR consisted of 25 cycles of amplification with priming temperature and elongation time optimized for each amplicon multiplex. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase. .. For the simultaneous mutation scanning and genotyping procedure, the same conditions were used, except that (1) a primer asymmetry ratio of 1:5 (100 nM limiting primer, 500 nM excess primer) was used to favor the production of the DNA strand targeted by the probe, and (2) the unlabelled 3′ end-capped probe was included at 500 nM.

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: RecBK29Q CD and RecBC were obtained by transforming the RecBK29Q ATPase mutant pPB800 (a gift from S. Kowalczykowski) and pPB700 (a gift from P. Bianco) plasmids, respectively, into the RecBD-null V330 strain, and following the same purification protocol as for WT RecBCD. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Isolation:

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: Tick midguts were dissected, placed in RLT extraction buffer (Qiagen), and stored at −80°C until used for RNA isolation. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis.

Nickel Column:

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: All purification procedures were carried out at 4°C. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM). .. Fractions containing denatured RecD where loaded onto Superdex 200 equilibrated with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 6 M Urea.

Mouse Assay:

Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis
Article Snippet: Western blot analyses were performed according to standard procedures. .. AA6-treated/untreated 4T1-injected mice tumorigenic tissue (10–25 mg) and AA6-treated/untreated 4T1 cells samples were lysed in Laemmli buffer (Tris HCl 100 mM pH 6.8, SDS 4%, glycerol 20%, DTT 25 mM, NuPAGE LDS Sample Buffer 1x - Invitrogen). .. Nitrocellulose blotting membranes were probed with the following antibodies: ZEB-1 (Santa Cruz), CtBP-1 (Cell Signaling), GPNMB (Thermo Fisher Sc.), MMP-3 (BIOSS), SRC (Cell Signaling), KGDH (alias OGDH, Genetex), TET-1 (Genetex), TET-2 (Santa Cruz) and TET-3 (Novus Biologicals) flag (SIGMA), α-tubulin (Cell Signaling), GAPDH (abcam), Grb2 (Santa Cruz).

Dot Blot:

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: Paragraph title: Chromatin immunoprecipitation and dot blot assays ... Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min.

Polymerase Chain Reaction:

Article Title: Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme
Article Snippet: The primer sequences of each selected locus are displayed in Supplementary Table . .. All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample. .. An Applied Biosystems Thermal Cycler 2720 (Life Technologies, USA) was set up with a first cycle of denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 0.45 min, annealing at 58 °C for 0.45 min, elongation at 72 °C for 1 min, and a final extension step of 7 min at 72 °C.

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: The PCR consisted of 25 cycles of amplification with priming temperature and elongation time optimized for each amplicon multiplex. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase. .. For the simultaneous mutation scanning and genotyping procedure, the same conditions were used, except that (1) a primer asymmetry ratio of 1:5 (100 nM limiting primer, 500 nM excess primer) was used to favor the production of the DNA strand targeted by the probe, and (2) the unlabelled 3′ end-capped probe was included at 500 nM.

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: We performed the PCRs in a reaction mixture (10 µL) containing ca. .. 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The PCR cycling conditions were as follows: 10 min at 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C, and a final elongation for 5 min at 72 °C.

Article Title: Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions
Article Snippet: Then 1 μg of purified RNA was mixed with 10 μl of a solution consisting of a basic buffer (100 mM Tris-HCl, pH 8.3, containing 500 mM KCl and 15 mM MgCl2 Invitrogen, USA), dNTP (10mM; Fermentas, USA), random primers (Invitrogen, USA), OligoDT primers (Invitrogen, USA), RNaseOUT recombinant ribonuclease inhibitor (40 U μl−1 ; Invitrogen, USA), M-MLV reverse transcriptase (100 U μl−1 ). .. The reactions included master mix Syber Green (2×) (Applied Biosystems, USA) and 1 μl cDNA (1 μg) template and were run in triplicate on a real-time PCR system (StepOne; Applied Biosystems, USA).

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: The RecD gene flanked by HindIII and NdeI was obtained via PCR on the pPB800 plasmid with forward ( 5’-CTGATCGCATATGAAATTGCAAAAGCAATTACTGGAAGCTGTGGAG-3’ ) and backward ( 5’-GCTGACTAAAGCTTTTATTCCCGTGAACTAAACAACGCCGCCA-3’ ) primers (IDT), purified, cut and ligated into a HindIII and NdeI treated PET15b plasmid. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Protein Extraction:

Article Title: A proteomic landscape of diffuse-type gastric cancer
Article Snippet: Paragraph title: Protein extraction and trypsin digestion ... Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%).

Immunoprecipitation:

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min.

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). .. IgY antibodies previously raised against recombinant Cmr2 ( ) were used for co-IPs.

Bradford Protein Assay:

Article Title: A proteomic landscape of diffuse-type gastric cancer
Article Snippet: Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%). .. Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%).

Staining:

Article Title: Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones
Article Snippet: Tris-HCl buffer stock solution (1 M, pH 8.0) was purchased from Affymetrix (Santa Clara, CA, USA). .. Tris-HCl buffer stock solution (1 M, pH 8.0) was purchased from Affymetrix (Santa Clara, CA, USA).

Article Title: Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme
Article Snippet: All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample. .. All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample.

Agarose Gel Electrophoresis:

Article Title: Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme
Article Snippet: All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample. .. All amplifications were performed in a final volume of 50 µl consisting of 1X PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl) (Invitrogen, USA), 2.5 mM MgCl2 (Invitrogen, USA), 0.4 µM of each primer (Sigma-Aldrich, Germany), 200 µM dNTPs (Sigma-Aldrich, Germany), 1.25 U of Taq DNA polymerase (Invitrogen, USA), and 10 µl of treated sample.

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Nuclei pellets were incubated in nuclear lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA with 1× Halt protease inhibitor cocktail) for 30 min, further lysed with a syringe, and sonicated with Covaris S2 to obtain fragments between 400 and 1000 base pairs.

Silver Staining:

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). .. Co-IPs were carried out as previously described ( ) with the following modifications: Each immunoprecipitation (30 µL of resin) used S20 cell extract containing 2 mg of protein.

Chromatin Immunoprecipitation:

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: 20×106 cells were trypsinized and crosslinked with 1% paraformaldehyde (w/v) (ThermoFisher Scientific) at room temperature for 5 min, followed by 125 mM glycine (Sigma) for 5 min to quench the crosslinking and washed (cold 1× PBS, 1 mM PMSF). .. Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Nuclei pellets were incubated in nuclear lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA with 1× Halt protease inhibitor cocktail) for 30 min, further lysed with a syringe, and sonicated with Covaris S2 to obtain fragments between 400 and 1000 base pairs.

SDS Page:

Article Title: IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System
Article Snippet: Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL). .. Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL).

Article Title: Transfection of microRNA Mimics Should Be Used with Caution
Article Snippet: Data were analyzed using the ImageQuant software. .. HeLa cells were lysed in 1% NP-40 lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, 1 mM DTT and proteinase inhibitors (halt inhibitor, Thermo #78442), and subjected to separation on 8 or 10% SDS-PAGE gels and Western blot (10 μg whole cell lysate per lane). .. Antibodies used for Western blot were anti-Bim (Cell Signaling, 2933), anti-Pten (Cell Signaling, 9559), anti-Phlpp2 (Bethyl, A300-661A-1), and anti-Ship1 (Cell Signaling, 2728).

Article Title: Co-operative binding assay for the characterization of mGlu4 allosteric modulators
Article Snippet: The SDS PAGE was done using 20 μg of protein sample and the proteins were transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). .. After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with antibodies against mGlu4 (1:2000) at 4 °C for 12 h. Membranes were washed three times for 10 min and incubated with a secondary antibody (1:5000 dilution) ECL Anti-rabbit IgG (horseradish peroxidase linked whole antibody from donkey NA934V (GE Healthcare UK)) for 1 h. Blots were washed with TBST three times and developed (#34080 Thermo Supersignal West Pico Chemiluminescent substrate) on HyBlot CL Autoradiography Film (Cat No.: E3012 Denvill Scientific Inc.).

Plasmid Preparation:

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The PCR cycling conditions were as follows: 10 min at 94 °C, 38 cycles of 30 s at 94 °C, 30 s at 55 °C, and 45 s at 72 °C, and a final elongation for 5 min at 72 °C.

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: The RecD gene flanked by HindIII and NdeI was obtained via PCR on the pPB800 plasmid with forward ( 5’-CTGATCGCATATGAAATTGCAAAAGCAATTACTGGAAGCTGTGGAG-3’ ) and backward ( 5’-GCTGACTAAAGCTTTTATTCCCGTGAACTAAACAACGCCGCCA-3’ ) primers (IDT), purified, cut and ligated into a HindIII and NdeI treated PET15b plasmid. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Co-Immunoprecipitation Assay:

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Paragraph title: P. furiosus cell extract preparation and coimmunoprecipitation (co-IP) reactions ... Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells).

Selection:

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions. .. The PCR products were transformed into DH5α cells (Promega, Madison, WI, USA) after ligation into the pGEM T-Easy Vector (Promega).

Titration:

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: Concentrations of WGA DNAs were measured by standard picogreen titration. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase.

In Vitro:

Article Title: Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members
Article Snippet: This problem was avoided by storing them in concentrated aliquots at −80°C. .. The LNAzyme digestion buffer contained 100 mM NaCl, 50 mM RNase-free Tris (pH 8.0) (Ambion, Austin, TX), 10 mM LiCl, 1 μM LNAzyme, and 5 μl of an in vitro transcription reaction mixture containing the transcribed RNA (see above). .. All reagents except Tris were treated with 0.1% (vol/vol) diethylpyrocarbonate (DEPC) to inactivate nucleases.

Radio Immunoprecipitation:

Article Title: The functional significance of microRNA-145 in prostate cancer
Article Snippet: Stained cells were immediately analysed by flow cytometry (Cell Lab Quanta SC; Beckman Coulter). .. Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing 1 × protease inhibitor cocktail (Roche, Basel, Switzerland). .. Protein estimations were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions.

Random Hexamer Labeling:

Article Title: Rare, Evolutionarily Unlikely Missense Substitutions in ATM Confer Increased Risk of Breast Cancer
Article Snippet: Fifty nanograms of genomic DNA and 9 μl of a sample buffer containing random hexamer primers were heat denatured and cooled, allowing random priming of the hexamers, then 9 μl of reaction buffer and 1 μl of Phi29 DNA polymerase were added and incubated overnight at 30°C for linear DNA synthesis. .. For standard HRM mutation scanning, simplex secondary PCRs (PCR2) were then performed in 6 μl reaction volume containing 1.5 μl of 1:100 diluted PCR1 product, 1X Invitrogen PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 1.5 mM MgCl2, 500 nM dNTP, 400 nM forward and reverse primers, 0.5X LCGreen Plus (Idaho Technology), and 0.04 U/μL of Platinum Taq Polymerase.

Concentration Assay:

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: RecBC concentration was determined using εex,coeff . of 3.7 × 105 M−1 cm−1 . .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM).

Immu-Puri:

Article Title: miR-128 inhibits telomerase activity by targeting TERT mRNA
Article Snippet: Immunopurification of Argonaute from HeLa and Tera cell extracts was performed using the 4F9 antibody (4F9, Santa Cruz Biotechnology) as described previously [ ]. .. Protein concentration was adjusted across samples with buffer B [20 mM Tris-HCl pH 8.0, 140 mM KCl, 5 mM EDTA pH 8.0, 0.5% NP-40, 0.1% deoxycholate, 100 U/ml Rnaseout (ThermoFisher Scientific), 1 mM DTT and 1X Halt protease inhibitor cocktail (Pierce)].

Lysis:

Article Title: Local enrichment of HP1alpha at telomeres alters their structure and regulation of telomere protection
Article Snippet: 20×106 cells were trypsinized and crosslinked with 1% paraformaldehyde (w/v) (ThermoFisher Scientific) at room temperature for 5 min, followed by 125 mM glycine (Sigma) for 5 min to quench the crosslinking and washed (cold 1× PBS, 1 mM PMSF). .. Cells were resuspended into ChIP lysis buffer (0.5% NP-40, 85 mM KCl, 20 mM Tris-HCl pH 8.0 with 1× Halt protease inhibitor cocktail (ThermoFisher Scientific)) for 15 min, homogenized with a pellet pestle (ThermoFisher Scientific), and spun at 450 x g for 5 min. .. Nuclei pellets were incubated in nuclear lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA with 1× Halt protease inhibitor cocktail) for 30 min, further lysed with a syringe, and sonicated with Covaris S2 to obtain fragments between 400 and 1000 base pairs.

Article Title: IFN-λ Inhibits MiR-122 Transcription through a Stat3-HNF4α Inflammatory Feedback Loop in an IFN-α Resistant HCV Cell Culture System
Article Snippet: Cells were harvested by the treatment of trypsin-EDTA. .. Cells were lysed in ice-cold lysis buffer (50mM Tris HCl pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40 with complete protease inhibitor from Invitrogen) for 10 minutes in ice (about 1X106 cells/200 μL). .. Whole cells and cell debris were pelleted by low speed centrifugation and cleared supernatants were transferred to a new tube.

Article Title: Transfection of microRNA Mimics Should Be Used with Caution
Article Snippet: Data were analyzed using the ImageQuant software. .. HeLa cells were lysed in 1% NP-40 lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, 1 mM DTT and proteinase inhibitors (halt inhibitor, Thermo #78442), and subjected to separation on 8 or 10% SDS-PAGE gels and Western blot (10 μg whole cell lysate per lane). .. Antibodies used for Western blot were anti-Bim (Cell Signaling, 2933), anti-Pten (Cell Signaling, 9559), anti-Phlpp2 (Bethyl, A300-661A-1), and anti-Ship1 (Cell Signaling, 2728).

Article Title: A proteomic landscape of diffuse-type gastric cancer
Article Snippet: Specimen in which EBER nuclear expression was observed in > 20% of the tumor cells were considered EBER positive. .. Samples were minced and lysed in lysis buffer (8 M Urea, 100 mM Tris Hydrochloride, pH 8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min of sonication (3 s on and 3 s off, amplitude 25%). .. The lysate was centrifuged at 14,000×g for 10 min and the supernatant was collected as whole tissue extract.

Article Title: RNA-Seq Analysis of the Host Response to Staphylococcus aureus Skin and Soft Tissue Infection in a Mouse Model
Article Snippet: The processed gene expression data have been submitted to the NCBI Gene Expression Omnibus ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number GSE56227. .. Ears were harvested as indicated above and placed into 1 mL of lysis buffer (5 mM Tris pH 8, 150 mM NaCl, 1% NP-40 [Surfact-Amps, ThermoFisher], 1x protease inhibitor cocktail [Halt, ThermoFisher]) on ice, and then homogenized. .. Ear homogenates were centrifuged at 12000 × g for 10 minutes.

Article Title: Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system
Article Snippet: Cultures were grown to late log phase, harvested by centrifugation, and weighed. .. Lysis buffer (50 mM Tris-Cl at pH 8, 30 U/mL SUPERase• In [Life Technologies], 1× Complete-mini EDTA-free protease inhibitor [Roche]) was added (3 mL per 1 g of cells). .. The soluble fraction was collected as S20 cell extract, with protein quantified by Qubit assay (Life Technologies).

Article Title: A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion
Article Snippet: Tick midguts were dissected, placed in RLT extraction buffer (Qiagen), and stored at −80°C until used for RNA isolation. .. Alternatively, midguts were dissected in 1× PBS, homogenized in 100 μl of NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, HALT protease inhibitor cocktail [Thermo Scientific]), and stored at 4°C until used for protein analysis. .. Quantitative RT-PCR was used to determine transcript and rickettsial abundance as described above.

Article Title: Synergy between RecBCD subunits is essential for efficient DNA unwinding
Article Snippet: All purification procedures were carried out at 4°C. .. Similar to the purification from inclusion bodies by , eight liters of E.coli cells expressing Histidine tagged RecD (RecD for short) were lysed in Lysis Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 1 mM Benzamidine and 1 mM PMSF) using the microfluidizer, and centrifuged for 10 min at 7000 × g. Pellets containing RecD in inclusion bodies were suspended in Resuspension Buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 6 M Guanidinium chloride, 5 mM Imidazole) and centrifuged for 45 min at 50,000 × g. The supernatant was applied to a pre-equilibrated (Equilibration buffer: Tris-HCl pH 7.5, 500 mM NaCl, 8 M Urea and 5 mM Imidazole) Nickel column (2 ml of HisPur, Thermo), and eluted with a stepwise gradient of imidazole (25–300 mM). .. Fractions containing denatured RecD where loaded onto Superdex 200 equilibrated with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 6 M Urea.

Article Title: The N-Terminal Domain of cGAS Determines Preferential Association with Centromeric DNA and Innate Immune Activation in the Nucleus
Article Snippet: The recovered supernatant represented the cytosolic fraction and were stored on ice. .. Nuclei were lysed in 100μl of Lysis Buffer X (LBX) (50mM Tris pH 8.0, 2.5mM EDTA pH 8.0 (Invitrogen), 0.25% SDS (Euromedex)) in presence of cOmplete EDTA free Protease inhibitor cocktail (Roche). .. The lysates were sonicated for 20 minutes at 4°C in a Sonorex Digitec (model DT100) ultrasonic bath (Bandelin).

Fluorescence In Situ Hybridization:

Article Title: Identification of Causative Ciguatoxins in Red Snappers Lutjanus bohar Implicated in Ciguatera Fish Poisonings in Vietnam
Article Snippet: Whole genomic DNA was extracted from fish flesh by use of the QuickGene DNA tissue kit S (FUJIFILM, Tokyo, Japan), according to the manufacturer’s instructions. .. 50 ng of template DNA, 0.2 mM of each dNTP, 0.5 μM of each designed primer pair, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 , 0.01% w/v gelatin, Applied Biosystems, Foster City, CA, USA), and 0.25 U of Ampli Taq Gold (Applied Biosystems) on a thermal cycler (PC-808, ASTEC, Fukuoka, Japan) to amplify both regions.

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    Thermo Fisher t4 polynucleotide kinase
    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using <t>T4</t> polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).
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    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Journal: Nature

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    doi: 10.1038/s41586-018-0557-5

    Figure Lengend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Article Snippet: For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Techniques: Thin Layer Chromatography, Generated, Incubation

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the  rrnE  operon, which is representative of all seven rRNA operons in  E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF  strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Techniques: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis