Biomod/2014/Sendai/temp/0829/Experiment

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<body>

Experiment

The <a href="/wiki/Biomod/2014/Sendai/temp/0829/Simulation">simulation</a> proves that our system will work properly. As a next step, we would like to conduct experiments to verify the function of our system.
We have prepared all the materials to begin the experiments of the enzyme system. DNA strands and enzymes are carefully designed and chosen, respectively, both of which have already arrived.


DNA preparation

All DNAs for experiments are shown in Fig.1.

<img src="http://openwetware.org/images/5/5d/Experiment2014sendai.jpg" width="980" height="358">
Fig.1 DNAs for experiments


Following Table.1 shows the sequences of the above DNAs. We designed these sequences carefully to avoid unexpected cleaving by restriction enzymes.


Table.1 Sequences for experiment

Input 1 CTAGAACATACGCATTTGATTCGCGAATTCAGCAGTACTATTCAAGCGACGGCT
Input 2 CTAGAACATACGCATTTGATTCGCCCATGGAGCAGTACTATTCAAGCGACGGCT
Input 3 CTAGAACATACGCATTTGATTCGCAGATCTAGCAGTACTATTCAAGCGACGGCT
Template A TCACTTCTGCATCGATCACGACTAGCCGTCGCTTGAATAGTGTTA
Gate A + AGCGTCATCGGTCTTTAAGCAGTACTATTCAAGCGACGGCTTCACTTCTGC
Gate A - 1 TCACGACTCAGCCGTCGCTTGAATAGTACTGCTGAATTCCCGATGACGCT
Gate A - 2 TCACGACTCAGCCGTCGCTTGAATAGTACTGCTCCATGGCCGATGACGCT
Gate A - 3 TCACGACTCAGCCGTCGCTTGAATAGTACTGCTAGATCTCCGATGACGCT
Output A TAGGTACGCTCCGACAAGGT
Anchor ACCTTGTCGGAGCGTACCTATCACTTCTGCATCGA
Key-DNA(A1) TCGATGCAGAAGTGA


Enzyme preparation

Our system involves three types of enzymes, polymerase, nickase, and restriction enzyme. Thus all these enzymes need to work properly in the same buffer and temperature.
We decided to use the same kind of polymerase and nickase as in the paper[1] that deals with both polymerase and nickase in NEB buffer. We chose three restriction enzymes which have high activity rates in NEB buffer as candidates in our system. They are Eco-RI, Ncc-I, and Bgl-II.
All the input and gate A - DNAs are designed to have the same sequences except for the recognition sites for the restriction enzymes.
[1] Kevin Montagne et al. 2010



We are about to begin these experiments!

<img src="http://openwetware.org/images/d/df/Underconstruction.jpg" width="980px" height="653px">
Fig.2 The preparation for experiment has been completely finished!


(C)Copyright Biomod 2014 Team Sendai
E-MAIL:<a href="mailto:teamsendai2014@gmail.com">teamsendai2014@gmail.com</a>

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