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bu 107 kcl 1  (Jena Bioscience)


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    Jena Bioscience bu 107 kcl 1
    Bu 107 Kcl 1, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/bu+107+kcl+1/Potassium+Acetate+Solution+3+M+pH+5%2E2/pm40938744-51-86-112
    Average 90 stars, based on 3 article reviews
    bu 107 kcl 1 - by Bioz Stars, 2026-09
    90/100 stars

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    Saline:

    Article Title: Protocol for monitoring mRNA translation and degradation in human cell-free lysates.
    Article Snippet: Biological samples HeLa S3 lysate Custom preparation Prepared as described in the Star Protocol Chemicals, peptides, and recombinant proteins [α-32P]dCTP Hartmann Analytic Cat. No. SCP-205 Citrate buffer 1 mM, pH 6.5 Invitrogen Cat. No. AM7001 CleanCap reagent AG TriLink BioTechnologies Cat. No. N-7113-5 CloneAmp HiFi premix Takara Cat. No. 639298 Creatine kinase Roche Cat. No. 10127566001 Creatine phosphate Roche Cat. No. 10621714001 CutSmart buffer 103 New England Biolabs Cat. No. B6004S Cycloheximide Fluorochem CAS. .. No. 66-81-9 Cat. No. M01527 DL-dithiothreitol (DTT) 1 M in H2O Merck Cat. No. 43816-10ml DMEM Gibco Cat. No. 41966-029 EDTA Sigma-Aldrich Cat. No. E5134-500G Fetal calf serum BioConcept Cat. No. 2-01F30-I Formaldehyde loading dye (NorthernMax) Invitrogen Cat. No. AM8552 Gel loading dye, purple (63) New England Biolabs Cat. No. B7024S HEPES 1 M, pH 7.3 Fisher Bioreagents Cat. No. BP299-100 HindIII-HF New England Biolabs Cat. No. R3104 Inorganic pyrophosphatase (PPase) (0.1 U/μL) Thermo Scientific REF: EF0221 K-acetate 3 M, pH 5.3 Jena Bioscience Cat. No. BU-107 KCl 1 M Thermo scientific Cat. No. J62422.AK LDS sample loading buffer 43 Invitrogen Cat. No. NP0008 MES Sigma Cat. No. M3671-250G MgCl2 1 M Jena Bioscience Cat. No. BU-110-1M Murine RNase inhibitor Vazyme Cat. No. R301-01-AA NaCl 5 M in H2O Sigma Cat. No. S5150-1L Phosphate-buffered saline (PBS), 13, pH 7.4 Gibco Cat. No. 10010-031 Protease inhibitor cocktail 1003 Bimake Cat. No. B14002 Puromycin Santa Cruz Biotechnology Cat. No. sc-108071 rATP (100 mM) Thermo Scientific REF: R0441 rCTP (100 mM) Thermo Scientific REF: R0451 rGTP (100 mM) Thermo Scientific REF: R0461 rUTP (100 mM) Thermo Scientific REF: R0471 RNase-free water Sigma Cat. No. W4502-1L SDS 10% Fisher Bioreagents Cat. No. BP2436-1 T7 RNA polymerase Thermo Scientific REF: EP0111 T7 transcription buffer 53 Thermo Scientific REF: EP0111 Tris base Fisher Bioreagents Cat. No. BP152.5 Tris-HCl pH 7.5 Fisher Bioreagent Cat. No. BP1756-100 Trypan blue stain Gibco Cat. No. 15250-061 Trypsin-EDTA PBS BioConcept Cat. No. 5-51F00-H Turbo DNase (2 U/μL) Thermo Scientific REF: AM2238 ULTRAhyb ultrasensitive hybridization buffer Invitrogen Cat. No. AM8669 Critical commercial assays Renilla-Glo luciferase assay system Promega Cat. No. E2720 Maxwell RSC simplyRNA cells kit Promega Cat. No. AS1390 Monarch RNA cleanup kit New England Biolabs Cat. No. T2040L (Continued on next page) STAR Protocols 6, 104073, September 19, 2025 3 MATERIALS AND EQUIPMENT .. ChIP DNA Clean & Concentrator kit Zymo Research Cat. No. D5205 Vaccinia capping system New England Biolabs Cat. No. M2080S DecaLabel DNA labeling kit Thermo Scientific Cat. No. K0622 Amersham MicroSpin G-50 columns Cytiva Cat. No. 27533001 Experimental models: Cell lines HeLa S3 cells ATCC Cat. No. CCL-2.2 Oligonucleotides p200 5′UTR hRLuc for northern blotting probe preparation 5′-TCTGCAGAATTCGCCCTTCATG-3’ 5′-GCACGTTCATTTGCTTGCA-3′ Recombinant DNA p200: pCRII-RLuc-SV40-6xMS2-A80 Described in Karousis et al.5 N/A Software and algorithms GraphPad Prism GraphPad Software Version 10.0.2 https://www. graphpad.com/updates/ prism-1002-release-notes ImageJ NIH Version 1.52 https://imagej.net/ ij/download.html Excel Microsoft Version 2403 https://www. microsoft.com/en-us/microsoft365/download-office Other 2 L shaker flask – Nalgene Erlenmeyer PP Nalgene Cat. No. 4102-2000 250 mL shaker flask – Erlenmeyer PC, non-treated Avantor Cat. No. 214-0449 125 mL shaker flask – Erlenmeyer PC Corning Cat. No. 430421 75 cm2 or 150 cm2 cell culture flask, surface treated TPP Cat. No. 90076 or 90151 Dual centrifuge ZentriMix 380 R Hettich AG N/A 2 mL screw cap microtubes for dual centrifuge Sarstedt Cat. No. 72.693 White-bottom 96-well plate Greiner Cat. No. 655073 Microplate reader Promega GloMax Explorer or similar GM3500 UV irradiation chamber for crosslinking Stratalinker or similar N/A Nylon transfer membrane Cytiva, Amersham RPN303B Translation Buffer (TB) for lysate preparation Reagent stock Final concentration Amount (μL) HEPES 1M, pH 7.3 33.78 mM 33.8 K-acetate 3M, pH 5.3 63.06 mM 21.0 MgCl2 25 mM 0.68 mM 27.0 KCl 1M 54.05 mM 54.1 Creatine phosphate (500 mM) 13.51 mM 27.0 Creatine kinase (5 μg/ml) 230 ng/ml 45.9 Protease inhibitor cocktail 1003 13 10.0 MilliQ water to 1000 μL 781.1 Total – 1000 = 1 mL Store at 4◦C for not more than 6 h. 4 STAR Protocols 6, 104073, September 19, 2025

    Protease Inhibitor:

    Article Title: Protocol for monitoring mRNA translation and degradation in human cell-free lysates.
    Article Snippet: Biological samples HeLa S3 lysate Custom preparation Prepared as described in the Star Protocol Chemicals, peptides, and recombinant proteins [α-32P]dCTP Hartmann Analytic Cat. No. SCP-205 Citrate buffer 1 mM, pH 6.5 Invitrogen Cat. No. AM7001 CleanCap reagent AG TriLink BioTechnologies Cat. No. N-7113-5 CloneAmp HiFi premix Takara Cat. No. 639298 Creatine kinase Roche Cat. No. 10127566001 Creatine phosphate Roche Cat. No. 10621714001 CutSmart buffer 103 New England Biolabs Cat. No. B6004S Cycloheximide Fluorochem CAS. .. No. 66-81-9 Cat. No. M01527 DL-dithiothreitol (DTT) 1 M in H2O Merck Cat. No. 43816-10ml DMEM Gibco Cat. No. 41966-029 EDTA Sigma-Aldrich Cat. No. E5134-500G Fetal calf serum BioConcept Cat. No. 2-01F30-I Formaldehyde loading dye (NorthernMax) Invitrogen Cat. No. AM8552 Gel loading dye, purple (63) New England Biolabs Cat. No. B7024S HEPES 1 M, pH 7.3 Fisher Bioreagents Cat. No. BP299-100 HindIII-HF New England Biolabs Cat. No. R3104 Inorganic pyrophosphatase (PPase) (0.1 U/μL) Thermo Scientific REF: EF0221 K-acetate 3 M, pH 5.3 Jena Bioscience Cat. No. BU-107 KCl 1 M Thermo scientific Cat. No. J62422.AK LDS sample loading buffer 43 Invitrogen Cat. No. NP0008 MES Sigma Cat. No. M3671-250G MgCl2 1 M Jena Bioscience Cat. No. BU-110-1M Murine RNase inhibitor Vazyme Cat. No. R301-01-AA NaCl 5 M in H2O Sigma Cat. No. S5150-1L Phosphate-buffered saline (PBS), 13, pH 7.4 Gibco Cat. No. 10010-031 Protease inhibitor cocktail 1003 Bimake Cat. No. B14002 Puromycin Santa Cruz Biotechnology Cat. No. sc-108071 rATP (100 mM) Thermo Scientific REF: R0441 rCTP (100 mM) Thermo Scientific REF: R0451 rGTP (100 mM) Thermo Scientific REF: R0461 rUTP (100 mM) Thermo Scientific REF: R0471 RNase-free water Sigma Cat. No. W4502-1L SDS 10% Fisher Bioreagents Cat. No. BP2436-1 T7 RNA polymerase Thermo Scientific REF: EP0111 T7 transcription buffer 53 Thermo Scientific REF: EP0111 Tris base Fisher Bioreagents Cat. No. BP152.5 Tris-HCl pH 7.5 Fisher Bioreagent Cat. No. BP1756-100 Trypan blue stain Gibco Cat. No. 15250-061 Trypsin-EDTA PBS BioConcept Cat. No. 5-51F00-H Turbo DNase (2 U/μL) Thermo Scientific REF: AM2238 ULTRAhyb ultrasensitive hybridization buffer Invitrogen Cat. No. AM8669 Critical commercial assays Renilla-Glo luciferase assay system Promega Cat. No. E2720 Maxwell RSC simplyRNA cells kit Promega Cat. No. AS1390 Monarch RNA cleanup kit New England Biolabs Cat. No. T2040L (Continued on next page) STAR Protocols 6, 104073, September 19, 2025 3 MATERIALS AND EQUIPMENT .. ChIP DNA Clean & Concentrator kit Zymo Research Cat. No. D5205 Vaccinia capping system New England Biolabs Cat. No. M2080S DecaLabel DNA labeling kit Thermo Scientific Cat. No. K0622 Amersham MicroSpin G-50 columns Cytiva Cat. No. 27533001 Experimental models: Cell lines HeLa S3 cells ATCC Cat. No. CCL-2.2 Oligonucleotides p200 5′UTR hRLuc for northern blotting probe preparation 5′-TCTGCAGAATTCGCCCTTCATG-3’ 5′-GCACGTTCATTTGCTTGCA-3′ Recombinant DNA p200: pCRII-RLuc-SV40-6xMS2-A80 Described in Karousis et al.5 N/A Software and algorithms GraphPad Prism GraphPad Software Version 10.0.2 https://www. graphpad.com/updates/ prism-1002-release-notes ImageJ NIH Version 1.52 https://imagej.net/ ij/download.html Excel Microsoft Version 2403 https://www. microsoft.com/en-us/microsoft365/download-office Other 2 L shaker flask – Nalgene Erlenmeyer PP Nalgene Cat. No. 4102-2000 250 mL shaker flask – Erlenmeyer PC, non-treated Avantor Cat. No. 214-0449 125 mL shaker flask – Erlenmeyer PC Corning Cat. No. 430421 75 cm2 or 150 cm2 cell culture flask, surface treated TPP Cat. No. 90076 or 90151 Dual centrifuge ZentriMix 380 R Hettich AG N/A 2 mL screw cap microtubes for dual centrifuge Sarstedt Cat. No. 72.693 White-bottom 96-well plate Greiner Cat. No. 655073 Microplate reader Promega GloMax Explorer or similar GM3500 UV irradiation chamber for crosslinking Stratalinker or similar N/A Nylon transfer membrane Cytiva, Amersham RPN303B Translation Buffer (TB) for lysate preparation Reagent stock Final concentration Amount (μL) HEPES 1M, pH 7.3 33.78 mM 33.8 K-acetate 3M, pH 5.3 63.06 mM 21.0 MgCl2 25 mM 0.68 mM 27.0 KCl 1M 54.05 mM 54.1 Creatine phosphate (500 mM) 13.51 mM 27.0 Creatine kinase (5 μg/ml) 230 ng/ml 45.9 Protease inhibitor cocktail 1003 13 10.0 MilliQ water to 1000 μL 781.1 Total – 1000 = 1 mL Store at 4◦C for not more than 6 h. 4 STAR Protocols 6, 104073, September 19, 2025

    Staining:

    Article Title: Protocol for monitoring mRNA translation and degradation in human cell-free lysates.
    Article Snippet: Biological samples HeLa S3 lysate Custom preparation Prepared as described in the Star Protocol Chemicals, peptides, and recombinant proteins [α-32P]dCTP Hartmann Analytic Cat. No. SCP-205 Citrate buffer 1 mM, pH 6.5 Invitrogen Cat. No. AM7001 CleanCap reagent AG TriLink BioTechnologies Cat. No. N-7113-5 CloneAmp HiFi premix Takara Cat. No. 639298 Creatine kinase Roche Cat. No. 10127566001 Creatine phosphate Roche Cat. No. 10621714001 CutSmart buffer 103 New England Biolabs Cat. No. B6004S Cycloheximide Fluorochem CAS. .. No. 66-81-9 Cat. No. M01527 DL-dithiothreitol (DTT) 1 M in H2O Merck Cat. No. 43816-10ml DMEM Gibco Cat. No. 41966-029 EDTA Sigma-Aldrich Cat. No. E5134-500G Fetal calf serum BioConcept Cat. No. 2-01F30-I Formaldehyde loading dye (NorthernMax) Invitrogen Cat. No. AM8552 Gel loading dye, purple (63) New England Biolabs Cat. No. B7024S HEPES 1 M, pH 7.3 Fisher Bioreagents Cat. No. BP299-100 HindIII-HF New England Biolabs Cat. No. R3104 Inorganic pyrophosphatase (PPase) (0.1 U/μL) Thermo Scientific REF: EF0221 K-acetate 3 M, pH 5.3 Jena Bioscience Cat. No. BU-107 KCl 1 M Thermo scientific Cat. No. J62422.AK LDS sample loading buffer 43 Invitrogen Cat. No. NP0008 MES Sigma Cat. No. M3671-250G MgCl2 1 M Jena Bioscience Cat. No. BU-110-1M Murine RNase inhibitor Vazyme Cat. No. R301-01-AA NaCl 5 M in H2O Sigma Cat. No. S5150-1L Phosphate-buffered saline (PBS), 13, pH 7.4 Gibco Cat. No. 10010-031 Protease inhibitor cocktail 1003 Bimake Cat. No. B14002 Puromycin Santa Cruz Biotechnology Cat. No. sc-108071 rATP (100 mM) Thermo Scientific REF: R0441 rCTP (100 mM) Thermo Scientific REF: R0451 rGTP (100 mM) Thermo Scientific REF: R0461 rUTP (100 mM) Thermo Scientific REF: R0471 RNase-free water Sigma Cat. No. W4502-1L SDS 10% Fisher Bioreagents Cat. No. BP2436-1 T7 RNA polymerase Thermo Scientific REF: EP0111 T7 transcription buffer 53 Thermo Scientific REF: EP0111 Tris base Fisher Bioreagents Cat. No. BP152.5 Tris-HCl pH 7.5 Fisher Bioreagent Cat. No. BP1756-100 Trypan blue stain Gibco Cat. No. 15250-061 Trypsin-EDTA PBS BioConcept Cat. No. 5-51F00-H Turbo DNase (2 U/μL) Thermo Scientific REF: AM2238 ULTRAhyb ultrasensitive hybridization buffer Invitrogen Cat. No. AM8669 Critical commercial assays Renilla-Glo luciferase assay system Promega Cat. No. E2720 Maxwell RSC simplyRNA cells kit Promega Cat. No. AS1390 Monarch RNA cleanup kit New England Biolabs Cat. No. T2040L (Continued on next page) STAR Protocols 6, 104073, September 19, 2025 3 MATERIALS AND EQUIPMENT .. ChIP DNA Clean & Concentrator kit Zymo Research Cat. No. D5205 Vaccinia capping system New England Biolabs Cat. No. M2080S DecaLabel DNA labeling kit Thermo Scientific Cat. No. K0622 Amersham MicroSpin G-50 columns Cytiva Cat. No. 27533001 Experimental models: Cell lines HeLa S3 cells ATCC Cat. No. CCL-2.2 Oligonucleotides p200 5′UTR hRLuc for northern blotting probe preparation 5′-TCTGCAGAATTCGCCCTTCATG-3’ 5′-GCACGTTCATTTGCTTGCA-3′ Recombinant DNA p200: pCRII-RLuc-SV40-6xMS2-A80 Described in Karousis et al.5 N/A Software and algorithms GraphPad Prism GraphPad Software Version 10.0.2 https://www. graphpad.com/updates/ prism-1002-release-notes ImageJ NIH Version 1.52 https://imagej.net/ ij/download.html Excel Microsoft Version 2403 https://www. microsoft.com/en-us/microsoft365/download-office Other 2 L shaker flask – Nalgene Erlenmeyer PP Nalgene Cat. No. 4102-2000 250 mL shaker flask – Erlenmeyer PC, non-treated Avantor Cat. No. 214-0449 125 mL shaker flask – Erlenmeyer PC Corning Cat. No. 430421 75 cm2 or 150 cm2 cell culture flask, surface treated TPP Cat. No. 90076 or 90151 Dual centrifuge ZentriMix 380 R Hettich AG N/A 2 mL screw cap microtubes for dual centrifuge Sarstedt Cat. No. 72.693 White-bottom 96-well plate Greiner Cat. No. 655073 Microplate reader Promega GloMax Explorer or similar GM3500 UV irradiation chamber for crosslinking Stratalinker or similar N/A Nylon transfer membrane Cytiva, Amersham RPN303B Translation Buffer (TB) for lysate preparation Reagent stock Final concentration Amount (μL) HEPES 1M, pH 7.3 33.78 mM 33.8 K-acetate 3M, pH 5.3 63.06 mM 21.0 MgCl2 25 mM 0.68 mM 27.0 KCl 1M 54.05 mM 54.1 Creatine phosphate (500 mM) 13.51 mM 27.0 Creatine kinase (5 μg/ml) 230 ng/ml 45.9 Protease inhibitor cocktail 1003 13 10.0 MilliQ water to 1000 μL 781.1 Total – 1000 = 1 mL Store at 4◦C for not more than 6 h. 4 STAR Protocols 6, 104073, September 19, 2025

    Hybridization:

    Article Title: Protocol for monitoring mRNA translation and degradation in human cell-free lysates.
    Article Snippet: Biological samples HeLa S3 lysate Custom preparation Prepared as described in the Star Protocol Chemicals, peptides, and recombinant proteins [α-32P]dCTP Hartmann Analytic Cat. No. SCP-205 Citrate buffer 1 mM, pH 6.5 Invitrogen Cat. No. AM7001 CleanCap reagent AG TriLink BioTechnologies Cat. No. N-7113-5 CloneAmp HiFi premix Takara Cat. No. 639298 Creatine kinase Roche Cat. No. 10127566001 Creatine phosphate Roche Cat. No. 10621714001 CutSmart buffer 103 New England Biolabs Cat. No. B6004S Cycloheximide Fluorochem CAS. .. No. 66-81-9 Cat. No. M01527 DL-dithiothreitol (DTT) 1 M in H2O Merck Cat. No. 43816-10ml DMEM Gibco Cat. No. 41966-029 EDTA Sigma-Aldrich Cat. No. E5134-500G Fetal calf serum BioConcept Cat. No. 2-01F30-I Formaldehyde loading dye (NorthernMax) Invitrogen Cat. No. AM8552 Gel loading dye, purple (63) New England Biolabs Cat. No. B7024S HEPES 1 M, pH 7.3 Fisher Bioreagents Cat. No. BP299-100 HindIII-HF New England Biolabs Cat. No. R3104 Inorganic pyrophosphatase (PPase) (0.1 U/μL) Thermo Scientific REF: EF0221 K-acetate 3 M, pH 5.3 Jena Bioscience Cat. No. BU-107 KCl 1 M Thermo scientific Cat. No. J62422.AK LDS sample loading buffer 43 Invitrogen Cat. No. NP0008 MES Sigma Cat. No. M3671-250G MgCl2 1 M Jena Bioscience Cat. No. BU-110-1M Murine RNase inhibitor Vazyme Cat. No. R301-01-AA NaCl 5 M in H2O Sigma Cat. No. S5150-1L Phosphate-buffered saline (PBS), 13, pH 7.4 Gibco Cat. No. 10010-031 Protease inhibitor cocktail 1003 Bimake Cat. No. B14002 Puromycin Santa Cruz Biotechnology Cat. No. sc-108071 rATP (100 mM) Thermo Scientific REF: R0441 rCTP (100 mM) Thermo Scientific REF: R0451 rGTP (100 mM) Thermo Scientific REF: R0461 rUTP (100 mM) Thermo Scientific REF: R0471 RNase-free water Sigma Cat. No. W4502-1L SDS 10% Fisher Bioreagents Cat. No. BP2436-1 T7 RNA polymerase Thermo Scientific REF: EP0111 T7 transcription buffer 53 Thermo Scientific REF: EP0111 Tris base Fisher Bioreagents Cat. No. BP152.5 Tris-HCl pH 7.5 Fisher Bioreagent Cat. No. BP1756-100 Trypan blue stain Gibco Cat. No. 15250-061 Trypsin-EDTA PBS BioConcept Cat. No. 5-51F00-H Turbo DNase (2 U/μL) Thermo Scientific REF: AM2238 ULTRAhyb ultrasensitive hybridization buffer Invitrogen Cat. No. AM8669 Critical commercial assays Renilla-Glo luciferase assay system Promega Cat. No. E2720 Maxwell RSC simplyRNA cells kit Promega Cat. No. AS1390 Monarch RNA cleanup kit New England Biolabs Cat. No. T2040L (Continued on next page) STAR Protocols 6, 104073, September 19, 2025 3 MATERIALS AND EQUIPMENT .. ChIP DNA Clean & Concentrator kit Zymo Research Cat. No. D5205 Vaccinia capping system New England Biolabs Cat. No. M2080S DecaLabel DNA labeling kit Thermo Scientific Cat. No. K0622 Amersham MicroSpin G-50 columns Cytiva Cat. No. 27533001 Experimental models: Cell lines HeLa S3 cells ATCC Cat. No. CCL-2.2 Oligonucleotides p200 5′UTR hRLuc for northern blotting probe preparation 5′-TCTGCAGAATTCGCCCTTCATG-3’ 5′-GCACGTTCATTTGCTTGCA-3′ Recombinant DNA p200: pCRII-RLuc-SV40-6xMS2-A80 Described in Karousis et al.5 N/A Software and algorithms GraphPad Prism GraphPad Software Version 10.0.2 https://www. graphpad.com/updates/ prism-1002-release-notes ImageJ NIH Version 1.52 https://imagej.net/ ij/download.html Excel Microsoft Version 2403 https://www. microsoft.com/en-us/microsoft365/download-office Other 2 L shaker flask – Nalgene Erlenmeyer PP Nalgene Cat. No. 4102-2000 250 mL shaker flask – Erlenmeyer PC, non-treated Avantor Cat. No. 214-0449 125 mL shaker flask – Erlenmeyer PC Corning Cat. No. 430421 75 cm2 or 150 cm2 cell culture flask, surface treated TPP Cat. No. 90076 or 90151 Dual centrifuge ZentriMix 380 R Hettich AG N/A 2 mL screw cap microtubes for dual centrifuge Sarstedt Cat. No. 72.693 White-bottom 96-well plate Greiner Cat. No. 655073 Microplate reader Promega GloMax Explorer or similar GM3500 UV irradiation chamber for crosslinking Stratalinker or similar N/A Nylon transfer membrane Cytiva, Amersham RPN303B Translation Buffer (TB) for lysate preparation Reagent stock Final concentration Amount (μL) HEPES 1M, pH 7.3 33.78 mM 33.8 K-acetate 3M, pH 5.3 63.06 mM 21.0 MgCl2 25 mM 0.68 mM 27.0 KCl 1M 54.05 mM 54.1 Creatine phosphate (500 mM) 13.51 mM 27.0 Creatine kinase (5 μg/ml) 230 ng/ml 45.9 Protease inhibitor cocktail 1003 13 10.0 MilliQ water to 1000 μL 781.1 Total – 1000 = 1 mL Store at 4◦C for not more than 6 h. 4 STAR Protocols 6, 104073, September 19, 2025

    Luciferase:

    Article Title: Protocol for monitoring mRNA translation and degradation in human cell-free lysates.
    Article Snippet: Biological samples HeLa S3 lysate Custom preparation Prepared as described in the Star Protocol Chemicals, peptides, and recombinant proteins [α-32P]dCTP Hartmann Analytic Cat. No. SCP-205 Citrate buffer 1 mM, pH 6.5 Invitrogen Cat. No. AM7001 CleanCap reagent AG TriLink BioTechnologies Cat. No. N-7113-5 CloneAmp HiFi premix Takara Cat. No. 639298 Creatine kinase Roche Cat. No. 10127566001 Creatine phosphate Roche Cat. No. 10621714001 CutSmart buffer 103 New England Biolabs Cat. No. B6004S Cycloheximide Fluorochem CAS. .. No. 66-81-9 Cat. No. M01527 DL-dithiothreitol (DTT) 1 M in H2O Merck Cat. No. 43816-10ml DMEM Gibco Cat. No. 41966-029 EDTA Sigma-Aldrich Cat. No. E5134-500G Fetal calf serum BioConcept Cat. No. 2-01F30-I Formaldehyde loading dye (NorthernMax) Invitrogen Cat. No. AM8552 Gel loading dye, purple (63) New England Biolabs Cat. No. B7024S HEPES 1 M, pH 7.3 Fisher Bioreagents Cat. No. BP299-100 HindIII-HF New England Biolabs Cat. No. R3104 Inorganic pyrophosphatase (PPase) (0.1 U/μL) Thermo Scientific REF: EF0221 K-acetate 3 M, pH 5.3 Jena Bioscience Cat. No. BU-107 KCl 1 M Thermo scientific Cat. No. J62422.AK LDS sample loading buffer 43 Invitrogen Cat. No. NP0008 MES Sigma Cat. No. M3671-250G MgCl2 1 M Jena Bioscience Cat. No. BU-110-1M Murine RNase inhibitor Vazyme Cat. No. R301-01-AA NaCl 5 M in H2O Sigma Cat. No. S5150-1L Phosphate-buffered saline (PBS), 13, pH 7.4 Gibco Cat. No. 10010-031 Protease inhibitor cocktail 1003 Bimake Cat. No. B14002 Puromycin Santa Cruz Biotechnology Cat. No. sc-108071 rATP (100 mM) Thermo Scientific REF: R0441 rCTP (100 mM) Thermo Scientific REF: R0451 rGTP (100 mM) Thermo Scientific REF: R0461 rUTP (100 mM) Thermo Scientific REF: R0471 RNase-free water Sigma Cat. No. W4502-1L SDS 10% Fisher Bioreagents Cat. No. BP2436-1 T7 RNA polymerase Thermo Scientific REF: EP0111 T7 transcription buffer 53 Thermo Scientific REF: EP0111 Tris base Fisher Bioreagents Cat. No. BP152.5 Tris-HCl pH 7.5 Fisher Bioreagent Cat. No. BP1756-100 Trypan blue stain Gibco Cat. No. 15250-061 Trypsin-EDTA PBS BioConcept Cat. No. 5-51F00-H Turbo DNase (2 U/μL) Thermo Scientific REF: AM2238 ULTRAhyb ultrasensitive hybridization buffer Invitrogen Cat. No. AM8669 Critical commercial assays Renilla-Glo luciferase assay system Promega Cat. No. E2720 Maxwell RSC simplyRNA cells kit Promega Cat. No. AS1390 Monarch RNA cleanup kit New England Biolabs Cat. No. T2040L (Continued on next page) STAR Protocols 6, 104073, September 19, 2025 3 MATERIALS AND EQUIPMENT .. ChIP DNA Clean & Concentrator kit Zymo Research Cat. No. D5205 Vaccinia capping system New England Biolabs Cat. No. M2080S DecaLabel DNA labeling kit Thermo Scientific Cat. No. K0622 Amersham MicroSpin G-50 columns Cytiva Cat. No. 27533001 Experimental models: Cell lines HeLa S3 cells ATCC Cat. No. CCL-2.2 Oligonucleotides p200 5′UTR hRLuc for northern blotting probe preparation 5′-TCTGCAGAATTCGCCCTTCATG-3’ 5′-GCACGTTCATTTGCTTGCA-3′ Recombinant DNA p200: pCRII-RLuc-SV40-6xMS2-A80 Described in Karousis et al.5 N/A Software and algorithms GraphPad Prism GraphPad Software Version 10.0.2 https://www. graphpad.com/updates/ prism-1002-release-notes ImageJ NIH Version 1.52 https://imagej.net/ ij/download.html Excel Microsoft Version 2403 https://www. microsoft.com/en-us/microsoft365/download-office Other 2 L shaker flask – Nalgene Erlenmeyer PP Nalgene Cat. No. 4102-2000 250 mL shaker flask – Erlenmeyer PC, non-treated Avantor Cat. No. 214-0449 125 mL shaker flask – Erlenmeyer PC Corning Cat. No. 430421 75 cm2 or 150 cm2 cell culture flask, surface treated TPP Cat. No. 90076 or 90151 Dual centrifuge ZentriMix 380 R Hettich AG N/A 2 mL screw cap microtubes for dual centrifuge Sarstedt Cat. No. 72.693 White-bottom 96-well plate Greiner Cat. No. 655073 Microplate reader Promega GloMax Explorer or similar GM3500 UV irradiation chamber for crosslinking Stratalinker or similar N/A Nylon transfer membrane Cytiva, Amersham RPN303B Translation Buffer (TB) for lysate preparation Reagent stock Final concentration Amount (μL) HEPES 1M, pH 7.3 33.78 mM 33.8 K-acetate 3M, pH 5.3 63.06 mM 21.0 MgCl2 25 mM 0.68 mM 27.0 KCl 1M 54.05 mM 54.1 Creatine phosphate (500 mM) 13.51 mM 27.0 Creatine kinase (5 μg/ml) 230 ng/ml 45.9 Protease inhibitor cocktail 1003 13 10.0 MilliQ water to 1000 μL 781.1 Total – 1000 = 1 mL Store at 4◦C for not more than 6 h. 4 STAR Protocols 6, 104073, September 19, 2025



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    https://www.bioz.com/product/bu+107+kcl+1/Potassium+Acetate+Solution+3+M+pH+5%2E2/pm40938744-51-86-112
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