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Team:Warsaw/Calendar-Main/22 July 2009
From 2009.igem.org
(Difference between revisions)
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| - | + | <h3><div style="text-align: center;">Parts preparations</div></h3> | |
| + | <h4>Sebastian</h4> | ||
| + | <br/> | ||
| + | <p>Task 1:</p> | ||
| + | <ul> | ||
| + | <li>Digest of parts BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI</li> | ||
| + | <li>Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): 10ul of plasmid, 1ul of each enzyme, 5ul of Fermentas Tango Yellow buffer, 33ul H2O</li> | ||
| + | <li>Reaction mixture (BBa_B0032): 5ul of plasmid, 0,5ul of each enzyme, 2,5ul of Fermentas Tango Yellow buffer, 16ul H2O</li> | ||
| + | <li>Time of digest - 6h</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Task 2:</p> | ||
| + | <ul> | ||
| + | <li>Electrophoresis and gel-out</li> | ||
| + | </ul> | ||
| + | <br/> | ||
| + | <p>Methods:</p> | ||
| + | <ul> | ||
| + | <li>Electrophoresis of all samples volume</li> | ||
| + | <li>Order of samples: (1,2) BBa_E0032, (3,4) BBa_C0051, (5) DNA Ladder Mix, (6,8,9) BBa_C0040, (7) BBa_B0032</li> | ||
| + | <li>Gel-out of samples with A&A Biotechnology Kit</li> | ||
| + | </ul> | ||
Revision as of 11:32, 23 July 2009
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Assembly of invasion operon
Marcin
Task 1:
- Prepare chemocompetent bacteria
Detailed protocol
- Incubate 100 ml of liquid bacterial culture until the OD value is 0.55 or little more
- Incubate the bacteria on the ice for 15 minutes
- Centrifuge the bacteria at 4°C for 15 minutes
- Remove the supernatant and add 10 ml of 0.1 M solution of CaCl2
- Gently suspend the bacterial pellet and incubate the bacteria on the ice for 45 minutes
- Centrifuge the bacteria at 4°C for 15 minutes
- Remove the supernatant and add 2 to 5 ml of 0.1 M solution of CaCl2
- Prepate the aliquots of suspended bacteria which contain 100 μl of the solution
- Store the bacteria in -80°C
Assembly of endosomal detection operon
Marcin
Task 1:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Detailed protocol of ligation is described here. The only modification is total volume of ligation mixture prepared 20.07.09
- petri dish were hold in 37°C for 16 hours
Cloning of p53 coding sequence
Marcin
Task 1:
- Cloning p53 coding sequence to pKS plasmid
Methods:
- Ligation mixture composition: 10 μl digested p53, 25 μl digested pKS, 5 μl ligation buffer (Fermentas),8 μl MQ water, 2 μl ligase T4
- Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).
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 usage of half volume of ligation mixture prepared today/li>
Parts preparations
Sebastian
Task 1:
- Digest of parts BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032
Methods:
- Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI
- Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): 10ul of plasmid, 1ul of each enzyme, 5ul of Fermentas Tango Yellow buffer, 33ul H2O
- Reaction mixture (BBa_B0032): 5ul of plasmid, 0,5ul of each enzyme, 2,5ul of Fermentas Tango Yellow buffer, 16ul H2O
- Time of digest - 6h
Task 2:
- Electrophoresis and gel-out
Methods:
- Electrophoresis of all samples volume
- Order of samples: (1,2) BBa_E0032, (3,4) BBa_C0051, (5) DNA Ladder Mix, (6,8,9) BBa_C0040, (7) BBa_B0032
- Gel-out of samples with A&A Biotechnology Kit
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