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Team:Kyoto/Notebook
From 2012.igem.org
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==August 2== | ==August 2== | ||
| + | ==August 2== | ||
| + | '''Mutation of FT''' | ||
| + | {|class="wikitable" | ||
| + | |+Inverse PCR | ||
| + | !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 | ||
| + | |||
| + | {|class="wikitable" | ||
| + | |+Dpn1 Digestion | ||
| + | !PCR product!!Dpn1 | ||
| + | |- | ||
| + | |50||2 | ||
| + | |} 37°C,1h incubate | ||
| + | |||
| + | {|class="wikitable" | ||
| + | |+Self-ligation | ||
| + | !product!!MilliQ!!Ligase!!T4 Kinase!!Total | ||
| + | |- | ||
| + | |2||7||5||1||15 | ||
| + | |} | ||
| + | 16°C, 1h incubate | ||
| + | |||
| + | {|class="wikitable" | ||
| + | |+Transformation | ||
| + | !competent cell!!DNA | ||
| + | |- | ||
| + | |20||2 | ||
| + | |} | ||
| + | Cells were stored on ice for 30min. <br> | ||
| + | After 42°C 60sec heat shock, cells were stored on ice for 2min.<br> | ||
| + | Then cells were preincubated at 37°C for 1hr, plated to Kanamycin plate. | ||
| + | <br> | ||
| + | <br> | ||
==August 10== | ==August 10== | ||
Revision as of 14:08, 21 August 2012
August 2
August 2
Mutation of FT
| 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 preincubated at 37°C for 1hr, plated to Kanamycin plate.
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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