Difference between revisions of "Part:BBa K2973017:Experience"

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===Applications of BBa_K2973017===
 
===Applications of BBa_K2973017===
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In our project, we used the IS6110 gene as a DNA template, in order to perform isothermal amplification reactions with Recombinase Polymerase Amplification (RPA). Using this method, we were able to detect quantities as low as 10 to the minus 9 nanograms in reaction times ranging from five to 20 minutes. The primers used in the amplification reactions were BBa_K2973016 (forward) and BBa_K2973017 (reverse). These primers include 5' overhangs ( a T7 promoter and a trigger sequence) to allow for transcription by a T7 RNA Polymerase and translation by a toehold-switch regulated system encoding for a reporter gene (BBa_K2973007). The expected amplified product length is 136 bp.
 
  
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RPA reaction after clean up of the amplified product
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In our project, this primer (<partinfo>BBa_K2973017</partinfo>) was used along with its forward primer (<partinfo>BBa_K2973016</partinfo>) for the amplification reactions of the IS6110 gene (<partinfo>BBa_K2973009</partinfo>) that was used as a biomarker, in order to perform isothermal amplification reactions with Recombinase Polymerase Amplification (RPA). Using this method, we were able to detect quantities as low as 10 to the minus 9 nanograms in reaction times ranging from five to 20 minutes. These primers include 5' overhangs ( a T7 promoter and a trigger sequence) to allow for transcription by a T7 RNA Polymerase and translation by a toehold-switch regulated system encoding for a reporter gene (<partinfo>BBa_K2973007</partinfo>). The expected amplified product length is 136 bp.
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RPA reaction after clean up of the amplified product:
  
 
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       <img src="https://static.igem.org/mediawiki/parts/3/31/Thessaly2019_RPA_IS6110_Gel.jpeg" width="251"
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       <img src="https://2019.igem.org/wiki/images/7/7e/T--Thessaly--Registry_Figure6.png" width="502"
 
         height="225">
 
         height="225">
 
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PCR reaction
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PCR reaction:
  
 
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       <img src="https://static.igem.org/mediawiki/parts/e/e0/Thessaly_2019_PCR_IS6110_Gel.jpeg" width="285"
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       <img src="https://2019.igem.org/wiki/images/c/c7/T--Thessaly--Results_Figure3A.jpg" width="285"
 
         height="350">
 
         height="350">
 
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   </body>

Latest revision as of 13:02, 21 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2973017

In our project, this primer (BBa_K2973017) was used along with its forward primer (BBa_K2973016) for the amplification reactions of the IS6110 gene (BBa_K2973009) that was used as a biomarker, in order to perform isothermal amplification reactions with Recombinase Polymerase Amplification (RPA). Using this method, we were able to detect quantities as low as 10 to the minus 9 nanograms in reaction times ranging from five to 20 minutes. These primers include 5' overhangs ( a T7 promoter and a trigger sequence) to allow for transcription by a T7 RNA Polymerase and translation by a toehold-switch regulated system encoding for a reporter gene (BBa_K2973007). The expected amplified product length is 136 bp.


RPA reaction after clean up of the amplified product:

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PCR reaction:

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User Reviews

UNIQcf9fef880d4562b6-partinfo-00000006-QINU UNIQcf9fef880d4562b6-partinfo-00000007-QINU