Team:Chiba/Parts/DNA

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<h2 id="uptake" style="background-color:#ff9933"><center>DNA Biobrick</center></h2>
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<h3 style="background-color:#ffdead ">1.概要</h3>
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&nbsp;&nbsp;&nbsp;&nbsp;If you use pBAD promoter, you can regulate the expression and over expression. In order to combine the various parts, we improved BBa_I74608. We inserted BsaI site in both sides. Owing to this improvement, we were able to use Golden Gate. This part is designed BsaI site doesn’t remain on the vector after digesting BsaI. So, if you design insert so as not to remain BsaI site, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.<br>
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<h3 style="background-color:#ffdead ">2.実験</h3>
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内容
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We performed Golden Gate with this part (vector) and mRFP (insert) and checked the function. And we investigated the reaction rate changing mol ratio of vector to insert. The protocol is below.<br>
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1) PCR up insert with BsaI site<br>
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2) Golden Gate<br>
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3) transformation<br>
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Mixture list in Golden Gate is below.<br>
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-------------------------------------------<br>
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vector&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1    uL<br>
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insert&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1    uL<br>
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10x ligase buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3    uL<br>
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ligase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5 uL<br>
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BsaI&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.5 uL<br>
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NFW&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;13 uL<br>
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--------------------------------------------<br>
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total&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;15  uL<br>
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The concentration of vector and insert was prepared fixing the volume.<br>
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<h3 style="background-color:#ffdead ">3.結果</h3>
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内容
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Latest revision as of 08:40, 27 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

DNA Biobrick

1.概要

    If you use pBAD promoter, you can regulate the expression and over expression. In order to combine the various parts, we improved BBa_I74608. We inserted BsaI site in both sides. Owing to this improvement, we were able to use Golden Gate. This part is designed BsaI site doesn’t remain on the vector after digesting BsaI. So, if you design insert so as not to remain BsaI site, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.

2.実験

内容 We performed Golden Gate with this part (vector) and mRFP (insert) and checked the function. And we investigated the reaction rate changing mol ratio of vector to insert. The protocol is below.
1) PCR up insert with BsaI site
2) Golden Gate
3) transformation
Mixture list in Golden Gate is below.
-------------------------------------------
vector                        1 uL
insert                        1 uL
10x ligase buffer        3 uL
ligase                        1.5 uL
BsaI                       1.5 uL
NFW                       13 uL
--------------------------------------------
total                        15 uL
The concentration of vector and insert was prepared fixing the volume.

3.結果

内容

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