Difference between revisions of "Part:BBa K1614019:Experience"
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===Applications of BBa_K1614019===
 
===Applications of BBa_K1614019===
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ATP Aptamer JAWS1 Spinach 2.1 is a fusion of the BBa_K1330000 generated by DTU Danemark. According to our results, this fusion should contain the same properties as BBa_K1614014. Yet, this Spinach 2.1 is ligand dependend to ATP and should find its use in the detection of the small molecule ATP. Applications such as nucleotide sensing during in vitro transcription are possible. For more information check BBa_K1614014.
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==Measurement of the ATP Consumption during <i> in vitro </i> transcription <i>==
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===User Reviews===
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=Methods=
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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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==Results==
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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 <i> in vitro </i> transcription. Similar data could not be shown with setups not containing T7 RNA polymerase.  
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=User Reviews=
 
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Revision as of 16:24, 20 September 2015

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Applications of BBa_K1614019

Measurement of the ATP Consumption during in vitro transcription

Methods

To describe <i>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.

User Reviews

UNIQ32c5378de0f130b1-partinfo-00000000-QINU UNIQ32c5378de0f130b1-partinfo-00000001-QINU