Binding Assay:Article Title: Ribosome Profiling in the Model Diatom Thalassiosira pseudonana.
Article Snippet: .. REAGENTS AND SOLUTIONS Binding and washing buffer, 1× 5 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 M NaCl (Carl Roth, cat. no. 3957) Double the concentrations for a 2× buffer Store indefinitely at room temperature Denaturing sample loading buffer, 2× 98% (v/v) formamide (Carl Roth, cat. no. P040) 10 mM EDTA (Carl Roth, cat. no. 8043) 300 μg/ml bromophenol blue (Carl Roth, cat. no. A512) Store indefinitely at −20°C PAA-TBE gel, 7% Mix in a fume hood: 3.5 ml 30% acrylamide/bis-acrylamide solution (19:1) (BioRad, cat. no. 1610154) 1.5 ml 10× TBE (Promega, cat. no. V4251) 10 ml MilliQ H2O For polymerization add: 120 μl 10% APS (prepare fresh) (Carl Roth, cat. no. 9592) 12 μl TEMED (Carl Roth, cat. no. 2367) Mix gently and pour gel Insert well comb and allow 30 min for gel to polymerize before use Polysome resuspension buffer (PRB) 50 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 100 mM NH4Cl (Carl Roth, cat. no. K298) 200 mM sucrose (Carl Roth, cat. no. 9097) 10.5 mM magnesium acetate (Carl Roth, cat. no. P026) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 mM DTT (Carl Roth, cat. no. 6908) 0.01% cycloheximide (Carl Roth, cat. no. 8682) 0.1% Triton X-100 (Carl Roth, cat. no. 3051) 25 U/ml Turbo DNase (ThermoFisher Scientific, cat. no. AM2238 or AM2239) Mix all reagents in a fume hood Prepare fresh before every use and keep on ice RNA gel extraction buffer 300 mM NaOAc, pH 5.5 (ThermoFisher Scientific, cat. no. AM9740) 1 mM EDTA (Carl Roth, cat. no. 8043) 0.25% (w/v) SDS (Carl Roth, cat. no. CN30) Store indefinitely at room temperaturePichler et al. 18 of 25 Current Protocols 26911299, 2023, 7, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.843 by C ochrane Israel, W iley O nline L ibrary on [16/09/2023]. .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Sucrose chase solution 60 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 450 mM NaCl (Carl Roth, cat. no. 3957) 15 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 0.0001% Ponceau S (Carl Roth, cat. no. 5938) 60% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C 50 ml of solution are sufficient for four gradients Sucrose cushion solution 1 M sucrose (Carl Roth, cat. no. 9097) in PRB (see recipe) 20 U/ml SUPERase-In (ThermoFisher Scientific, cat. no. AM2694, AM2696) Prepare fresh before every use and keep on ice Sucrose solution, 10% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 10% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients Sucrose solution, 50% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 50% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients TBE loading buffer, 6× In 6.7 ml nuclease-free H2O, dissolve 25 mg bromophenol blue (Carl Roth, cat. no. A512) and 25 mg xylene cyanole (Carl Roth, cat. no. A513).
Hood:Article Title: Ribosome Profiling in the Model Diatom Thalassiosira pseudonana.
Article Snippet: .. REAGENTS AND SOLUTIONS Binding and washing buffer, 1× 5 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 M NaCl (Carl Roth, cat. no. 3957) Double the concentrations for a 2× buffer Store indefinitely at room temperature Denaturing sample loading buffer, 2× 98% (v/v) formamide (Carl Roth, cat. no. P040) 10 mM EDTA (Carl Roth, cat. no. 8043) 300 μg/ml bromophenol blue (Carl Roth, cat. no. A512) Store indefinitely at −20°C PAA-TBE gel, 7% Mix in a fume hood: 3.5 ml 30% acrylamide/bis-acrylamide solution (19:1) (BioRad, cat. no. 1610154) 1.5 ml 10× TBE (Promega, cat. no. V4251) 10 ml MilliQ H2O For polymerization add: 120 μl 10% APS (prepare fresh) (Carl Roth, cat. no. 9592) 12 μl TEMED (Carl Roth, cat. no. 2367) Mix gently and pour gel Insert well comb and allow 30 min for gel to polymerize before use Polysome resuspension buffer (PRB) 50 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 100 mM NH4Cl (Carl Roth, cat. no. K298) 200 mM sucrose (Carl Roth, cat. no. 9097) 10.5 mM magnesium acetate (Carl Roth, cat. no. P026) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 mM DTT (Carl Roth, cat. no. 6908) 0.01% cycloheximide (Carl Roth, cat. no. 8682) 0.1% Triton X-100 (Carl Roth, cat. no. 3051) 25 U/ml Turbo DNase (ThermoFisher Scientific, cat. no. AM2238 or AM2239) Mix all reagents in a fume hood Prepare fresh before every use and keep on ice RNA gel extraction buffer 300 mM NaOAc, pH 5.5 (ThermoFisher Scientific, cat. no. AM9740) 1 mM EDTA (Carl Roth, cat. no. 8043) 0.25% (w/v) SDS (Carl Roth, cat. no. CN30) Store indefinitely at room temperaturePichler et al. 18 of 25 Current Protocols 26911299, 2023, 7, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.843 by C ochrane Israel, W iley O nline L ibrary on [16/09/2023]. .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Sucrose chase solution 60 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 450 mM NaCl (Carl Roth, cat. no. 3957) 15 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 0.0001% Ponceau S (Carl Roth, cat. no. 5938) 60% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C 50 ml of solution are sufficient for four gradients Sucrose cushion solution 1 M sucrose (Carl Roth, cat. no. 9097) in PRB (see recipe) 20 U/ml SUPERase-In (ThermoFisher Scientific, cat. no. AM2694, AM2696) Prepare fresh before every use and keep on ice Sucrose solution, 10% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 10% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients Sucrose solution, 50% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 50% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients TBE loading buffer, 6× In 6.7 ml nuclease-free H2O, dissolve 25 mg bromophenol blue (Carl Roth, cat. no. A512) and 25 mg xylene cyanole (Carl Roth, cat. no. A513).
Gel Extraction:Article Title: Ribosome Profiling in the Model Diatom Thalassiosira pseudonana.
Article Snippet: .. REAGENTS AND SOLUTIONS Binding and washing buffer, 1× 5 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 M NaCl (Carl Roth, cat. no. 3957) Double the concentrations for a 2× buffer Store indefinitely at room temperature Denaturing sample loading buffer, 2× 98% (v/v) formamide (Carl Roth, cat. no. P040) 10 mM EDTA (Carl Roth, cat. no. 8043) 300 μg/ml bromophenol blue (Carl Roth, cat. no. A512) Store indefinitely at −20°C PAA-TBE gel, 7% Mix in a fume hood: 3.5 ml 30% acrylamide/bis-acrylamide solution (19:1) (BioRad, cat. no. 1610154) 1.5 ml 10× TBE (Promega, cat. no. V4251) 10 ml MilliQ H2O For polymerization add: 120 μl 10% APS (prepare fresh) (Carl Roth, cat. no. 9592) 12 μl TEMED (Carl Roth, cat. no. 2367) Mix gently and pour gel Insert well comb and allow 30 min for gel to polymerize before use Polysome resuspension buffer (PRB) 50 mM Tris·HCl, pH 8 (ThermoFisher Scientific, cat. no. 15568025) 100 mM NH4Cl (Carl Roth, cat. no. K298) 200 mM sucrose (Carl Roth, cat. no. 9097) 10.5 mM magnesium acetate (Carl Roth, cat. no. P026) 0.5 mM EDTA (Carl Roth, cat. no. 8043) 1 mM DTT (Carl Roth, cat. no. 6908) 0.01% cycloheximide (Carl Roth, cat. no. 8682) 0.1% Triton X-100 (Carl Roth, cat. no. 3051) 25 U/ml Turbo DNase (ThermoFisher Scientific, cat. no. AM2238 or AM2239) Mix all reagents in a fume hood Prepare fresh before every use and keep on ice RNA gel extraction buffer 300 mM NaOAc, pH 5.5 (ThermoFisher Scientific, cat. no. AM9740) 1 mM EDTA (Carl Roth, cat. no. 8043) 0.25% (w/v) SDS (Carl Roth, cat. no. CN30) Store indefinitely at room temperaturePichler et al. 18 of 25 Current Protocols 26911299, 2023, 7, D ow nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.843 by C ochrane Israel, W iley O nline L ibrary on [16/09/2023]. .. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Sucrose chase solution 60 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 450 mM NaCl (Carl Roth, cat. no. 3957) 15 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 0.0001% Ponceau S (Carl Roth, cat. no. 5938) 60% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C 50 ml of solution are sufficient for four gradients Sucrose cushion solution 1 M sucrose (Carl Roth, cat. no. 9097) in PRB (see recipe) 20 U/ml SUPERase-In (ThermoFisher Scientific, cat. no. AM2694, AM2696) Prepare fresh before every use and keep on ice Sucrose solution, 10% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 10% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients Sucrose solution, 50% 20 mM Tris·HCl, pH 7.4 (Carl Roth, cat. no. 4855) 150 mM NaCl (Carl Roth, cat. no. 3957) 5 mM MgCl2 (ThermoFisher Scientific, cat. no. R0971) 50% sucrose (w/v) (Carl Roth, cat. no. 9097) Store up to 3 days at 4°C Prior to use, add: 1 mM DTT (Carl Roth, cat. no. 6908) 100 μg/μl cycloheximide (Carl Roth, cat. no. 8682) 25 ml of solution is sufficient for four gradients TBE loading buffer, 6× In 6.7 ml nuclease-free H2O, dissolve 25 mg bromophenol blue (Carl Roth, cat. no. A512) and 25 mg xylene cyanole (Carl Roth, cat. no. A513).
other:
Article Title: Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae
Article Snippet: Hybridization took 375 on M ay 18, 2020 at B IO LO G IB IB LIO T E K E T http://iai.asm .org/ D ow nloaded from 15 place at 46°C (using an hybridization oven) and in 100 μl per well of hybridization buffer 376 (0.01% (v/v) SDS (Carl Roth, #CN30.2), 25% (v/v) formamide (Carl Roth, #P040.1), 900 mM 377 NaCl (Carl Roth, #9265.2), 20 mM Tris/HCl (pH 8.0) (Tris: Sigma-Aldrich, #B2261; HCl: 378 Sigma-Aldrich, #6331.3) in Milli-Q-water) containing the Cy5 labeled probe (1 pmol/μl).
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