Difference between revisions of "Part:BBa K1614019:Experience"
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===Applications of BBa_K1614019=== | ===Applications of BBa_K1614019=== | ||
| − | ==Measurement of the ATP Consumption during <i> in vitro </i> transcription | + | ==Measurement of the ATP Consumption during <i> in vitro </i> transcription== |
=Methods= | =Methods= | ||
To describe <i>in vitro</i> transcription time-resolved in terms of NTP consumption, following conditions were applied: 500 nM renatured ATP Aptamer Spinach2 RNA, 10 µM Malachite Green Aptamer DNA template, 40 mM Tris pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton X-100, 4 mM each NTP, 10 mM DTT, 5 % DMSO, 100 µM DFHBI, 1 mM malachite green, 0.1 mg/mL T7 RNA Polymerase, 0.2 U/µL Ribolock RNase and 0.1 U Pyrophosphatase (Thermo Scientific). The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2). | To describe <i>in vitro</i> transcription time-resolved in terms of NTP consumption, following conditions were applied: 500 nM renatured ATP Aptamer Spinach2 RNA, 10 µM Malachite Green Aptamer DNA template, 40 mM Tris pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton X-100, 4 mM each NTP, 10 mM DTT, 5 % DMSO, 100 µM DFHBI, 1 mM malachite green, 0.1 mg/mL T7 RNA Polymerase, 0.2 U/µL Ribolock RNase and 0.1 U Pyrophosphatase (Thermo Scientific). The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2). | ||
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Applications of BBa_K1614019
Measurement of the ATP Consumption during in vitro transcription
Methods
To describe in vitro transcription time-resolved in terms of NTP consumption, following conditions were applied: 500 nM renatured ATP Aptamer Spinach2 RNA, 10 µM Malachite Green Aptamer DNA template, 40 mM Tris pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton X-100, 4 mM each NTP, 10 mM DTT, 5 % DMSO, 100 µM DFHBI, 1 mM malachite green, 0.1 mg/mL T7 RNA Polymerase, 0.2 U/µL Ribolock RNase and 0.1 U Pyrophosphatase (Thermo Scientific). The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2).
Results
By adding the ATP Aptamer Spinach construct to a setup containg T7 RNA Polymerase and ATP we achieved time resolved data that show the decrease of fluorescence connected to the depletion of ATP during in vitro transcription. Similar data could not be shown with setups not containing T7 RNA polymerase.
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