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Team:Kyoto/Notebook
From 2012.igem.org
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==August 2== | ==August 2== | ||
| - | '''Mutation of FT''' | + | <small>by Sato</small><br> |
| + | '''Mutation of FT'''<br> | ||
| + | FT gene has two BioBrick restriction enzyme sites, EcoR1 and Pst1 which is next to each other.<br> | ||
| + | So we tried to delete both at once by using two primers with mutation.<br> | ||
{|class="wikitable" | {|class="wikitable" | ||
|+Inverse PCR | |+Inverse PCR | ||
| Line 32: | Line 35: | ||
Cells were stored on ice for 30min. <br> | Cells were stored on ice for 30min. <br> | ||
After 42°C 60sec heat shock, cells were stored on ice for 2min.<br> | After 42°C 60sec heat shock, cells were stored on ice for 2min.<br> | ||
| - | Then cells were | + | Then cells were precultureed at 37°C for 1hr, plated to Kanamycin plate. |
<br> | <br> | ||
<br> | <br> | ||
| + | ==August 13== | ||
| + | Liquid culture of FT 37°C, overnight | ||
| + | |||
| + | ==August 14== | ||
| + | =====Miniprep of FT===== | ||
| + | The concentrarion was 81.5ng/uL<br> | ||
| + | <br> | ||
| + | To check wheter mutation was succeed, | ||
| + | =====Restriction digestion and Electrophoresis=== | ||
==August 10== | ==August 10== | ||
Revision as of 15:32, 21 August 2012
Contents |
August 2
by Sato
Mutation of FT
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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