From 2008.igem.org
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| - | 3. Optimalization of conditions for <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C). <br> | + | 3. Optimization of conditions for <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C). <br> |
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| - | 4. Optimalization of conditions for <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA polymerase for transcription fusion; annealing temperature gradient (from 62°C to 82°C). <br> | + | 4. Optimization of conditions for <html><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - T7 RNA polymerase for transcription fusion; annealing temperature gradient (from 62°C to 82°C). <br> |
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| - | Template DNA: E. coli Rosetta and TOP10 (negative control) genomic DNA<br> | + | Template DNA: <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> (negative control) genomic DNA<br> |
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Revision as of 15:54, 1 October 2008
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Michał K.:
1.Setup of 9 separate cultures from 9 colonies of transformants pMPMT5+AID (liquid LB+tetracycline).
2. Optimization of conditions for PCR to obtain AID DNA fragment - annealing temperature gradient (from 62°C to 82°C).
Primers:
AIDlNrH
AIDpLinB
Template DNA: pTrc99A-AID
Elongation time: 60 sec
20 cycles
3. Optimization of conditions for PCR - T7 RNA polymerase for translation fusion; annealing temperature gradient (from 62°C to 82°C).
Primers:
T7lLinkB
T7pXbSal
Template DNA: E. coli Rosetta genomic DNA
Elongation time: 4 minutes
20 cycles
4. Optimization of conditions for PCR - T7 RNA polymerase for transcription fusion; annealing temperature gradient (from 62°C to 82°C).
Primers:
T7lRBSHi
T7pXbSal
Template DNA: E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> (negative control) genomic DNA
Elongation time: 4 minutes
20 cycles
5. Gel electrophoresis of PCR products.
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