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Team:Warsaw/Calendar-Main/17 July 2009
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(New page: {{WarNotebook}} <!-- do not edit above me! --> <html> <h3><div style="text-align: center;">Assembly of endosomal detection operon</div></h3> <h4>Marcin</h4> <p>Task 1:</p> <ul> <li>Ligat...) |
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| - | </ | + | <h3><div style="text-align: center;">Cloning of p53 coding sequence</div></h3> |
| + | <h4>Marcin</h4> | ||
| + | <p>Task:</p> | ||
| + | <ul> | ||
| + | <li>Isolation and evaluation of pKS/p53 plasmid</li> | ||
| + | </ul> | ||
| + | <b><p>Comment:</p></b> | ||
| + | <p>Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them.</p> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <ol> | ||
| + | <li>Prepare LB medium with kanamycin</li> | ||
| + | <li>Add 3.5 ml of the medium to the probes</li> | ||
| + | <li>Add one bacterial colony to each probe</li> | ||
| + | <li>Breed the bacteria about 8 hours</li> | ||
| + | </ol> | ||
| + | |||
| + | </html> | ||
Revision as of 23:38, 21 July 2009
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Assembly of endosomal detection operon
Marcin
Task 1:
- Ligations of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence I designed the following elemets (the order of biobricks is from the 5'end to 3' end):
- BBa_B0032+BBa_c0040
- BBa_B0032+BBa_C0051
- BBa_B0032+BBa_E0032
- BBa_B0032 (ligation of empty vector as a negative control of the experiment)
Methods:
- Ligation mixture composition: 3 μl digested plasmid with RBS, 3 μl digested CDS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 13 μl MQ water
- Negative control mixture composition: 3 μl digested plasmid with RBS, 5 μl ligation buffer (Fermentas, PEG4000 have been added previously), 1 μl ligase T4, 16 μl MQ water
- Duration of ligation was about 12 hours
Task 2:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of ligation is described here. The only modification is total volume of ligation mixture prepared 16.07.09
- petri dish were hold in 37°C for 36 hours
Cloning of p53 coding sequence
Marcin
Task:
- Isolation and evaluation of pKS/p53 plasmid
Comment:
Besides high effectivity of transformation all of the bacterial colonies were blue. It is possible that excess of X-Gal may cause some problems with the selection system. Due to this uncentainty I decide to prepare a few bacterial breedings to isolate the plasmids and digest them.
Methods:
- Prepare LB medium with kanamycin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 8 hours
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