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Team:Kyoto/Notebook
From 2012.igem.org
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To check wheter mutation was succeed, | To check wheter mutation was succeed, | ||
| - | =====Restriction digestion and Electrophoresis=== | + | =====Restriction digestion and Electrophoresis==== |
==August 10== | ==August 10== | ||
Revision as of 15:35, 21 August 2012
Contents |
August 2
Mutation of FT by Sato
FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.
So we tried to delete both at once by using two primers with mutation.
| 10xBufer | 2mM dNTPs | primer fwd | primer rev | template | polymerase | MilliQ | Total |
|---|---|---|---|---|---|---|---|
| 5 | 5 | 1.5 | 1.5 | 0.5 | 1 | 35.5 | 50 |
94°C 2min, (98°C 10sec, 68°C 4min)x2cycles, 4°C Hold
| PCR product | Dpn1 |
|---|---|
| 50 | 2 |
| product | MilliQ | Ligase | T4 Kinase | Total |
|---|---|---|---|---|
| 2 | 7 | 5 | 1 | 15 |
16°C, 1h incubate
| competent cell | DNA |
|---|---|
| 20 | 2 |
Cells were stored on ice for 30min.
After 42°C 60sec heat shock, cells were stored on ice for 2min.
Then cells were precultureed at 37°C for 1hr, plated to Kanamycin plate.
August 13
Liquid culture of FT 37°C, overnight
August 14
Miniprep of FT
The concentrarion was 81.5ng/uL
To check wheter mutation was succeed,
=Restriction digestion and Electrophoresis
August 10
Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.
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