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Team:Warsaw/Calendar-Main/25 July 2009
From 2009.igem.org
(Difference between revisions)
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<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
<li>Digest of isolate plasmids using XbaI and PstI</li> | <li>Digest of isolate plasmids using XbaI and PstI</li> | ||
| - | <ul><li>Reaction mixture composition: 0.5 μl purified plasmid DNA product | + | <ul><li>Reaction mixture composition: |
| + | <pre>0.5 μl purified plasmid DNA product | ||
| + | 0.5 μl XbaI (Fermentas) | ||
| + | 0.5 μl PstI (Fermentas) | ||
| + | 2 μl Buffer Tango (Fermentas) | ||
| + | 16.5 μl MQ water</pre></li></ul> | ||
<li>The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.</li> | <li>The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.</li> | ||
<li>In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li></ul> | <li>In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li></ul> | ||
| Line 34: | Line 39: | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
<li>Digest of BBa_B0032 using SpeI and PstI</li> | <li>Digest of BBa_B0032 using SpeI and PstI</li> | ||
| - | <ul><li>Reaction mixture composition: 10 μl purified plasmid DNA product | + | <ul><li>Reaction mixture composition: |
| + | <pre>10 μl purified plasmid DNA product | ||
| + | 0.5 μl SpeI (Fermentas) | ||
| + | 1 μl PstI (Fermentas) | ||
| + | 5 μl Buffer Tango (Fermentas) | ||
| + | 34 μl MQ water</pre></li></ul> | ||
<li>Digest of other biobricks using PstI and XbaI</li> | <li>Digest of other biobricks using PstI and XbaI</li> | ||
| - | <ul><li>Reaction mixture composition: 10 μl purified plasmid DNA product | + | <ul><li>Reaction mixture composition: |
| + | <pre>10 μl purified plasmid DNA product | ||
| + | 1 μl XbaI (Fermentas) | ||
| + | 1 μl PstI (Fermentas) | ||
| + | 5 μl Buffer Tango (Fermentas) | ||
| + | 34 μl MQ water</pre></li></ul> | ||
<li>Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes</li> | <li>Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes</li> | ||
</ul></li><br/> | </ul></li><br/> | ||
| Line 44: | Line 59: | ||
</ul> | </ul> | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
| - | <li>Ligation mixture composition: 8 μl digested plasmid with RBS | + | <li>Ligation mixture composition: |
| - | <li>Negative control mixture composition: 8 μl digested plasmid with RBS | + | <pre>8 μl digested plasmid with RBS |
| + | 9 μl digested CDS | ||
| + | 2 μl ligation buffer (Fermentas, PEG4000 have been added previously) | ||
| + | 1 μl ligase T4 (Fermentas)</pre></li> | ||
| + | <li>Negative control mixture composition: | ||
| + | 8 μl digested plasmid with RBS | ||
| + | 2 μl ligation buffer (Fermentas, PEG4000 have been added previously) | ||
| + | 1 μl ligase T4 (Fermentas) | ||
| + | 9 μl MQ water</pre></li> | ||
<li>Duration of ligation was about 12 hours</li> | <li>Duration of ligation was about 12 hours</li> | ||
</ul> | </ul> | ||
Latest revision as of 16:21, 26 September 2009
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Assembly of endosomal detection operon
Marcin
Task 1:
- Digest of isolate plasmids with ligated biobricks to verify the success of ligation
Methods:
- Digest of isolate plasmids using XbaI and PstI
- Reaction mixture composition:
0.5 μl purified plasmid DNA product 0.5 μl XbaI (Fermentas) 0.5 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 16.5 μl MQ water
- The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids
Comment:
All isolated plasmids have not insert. The cloning must be performed another time
Task 2:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:
Methods:
- Digest of BBa_B0032 using SpeI and PstI
- Reaction mixture composition:
10 μl purified plasmid DNA product 0.5 μl SpeI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 34 μl MQ water
- Digest of other biobricks using PstI and XbaI
- Reaction mixture composition:
10 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 34 μl MQ water
- Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80°C for 20 minutes
Task 3:
- Ligations of biobricks
Methods:
- Ligation mixture composition:
8 μl digested plasmid with RBS 9 μl digested CDS 2 μl ligation buffer (Fermentas, PEG4000 have been added previously) 1 μl ligase T4 (Fermentas)
- Negative control mixture composition: 8 μl digested plasmid with RBS 2 μl ligation buffer (Fermentas, PEG4000 have been added previously) 1 μl ligase T4 (Fermentas) 9 μl MQ water
- Duration of ligation was about 12 hours
Construction of K177012 operon1_part2
Ania
Tasks:
- Gel electrophoresis of the obtained clones.
- Repeat the ligation of the PcI and RBs.3LacI parts.
Results:
- Only small amount of DNA visible on the gel in 4 samples out of 20. Probably plasmid isolation has been done using too old solution 2. The visible DNA fragments are not the correct clone.
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