Team:Warsaw/Calendar-Main/1 October 2009
From 2009.igem.org
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Isolation of BBa_J63010 from 2009 Kit
Monika
Task:
- Isolate BioBrick BBa_J63010 from 2009 Kit Plate 1
Methods:
- Alkaline lysis with A&A Biotechnology Kit, procedure described here
- Control digestion of BBa_J63010
First reaction mixture composition:
5μl plasmid solution 0,3μl EcoRI (Fermentas) 0,3μl PstI (Fermentas) 2μl Buffer Orange (Fermentas) 12,4 μl MQ water
Second reaction mixture composition:
5μl plasmid solution 0,5μl PvuII (Fermentas) 2μl Buffer Green (Fermentas) 12,3 μl MQ water
Digestion in 37°C for 2,5h
Bistable switch testing
Monika
Tasks:
- Planting GFP expressing clones on LB medium with tetracycline
Cloning of the cro-box into the pSB1A3 plasmid
Kamil
Tasks:
- Plasmid isolation
- Control digest
Methods:
- The plasmids were isolated from 3μl of overnight liquid cultures using the Plasmid Mini kit (A&A Biotechnology).
- The digest mix was prepared as follows:
20μl purified plasmid
3μl Orange Buffer (Fermentas)
1μl PstI Enzyme (Fermentas)
1μl EcoRI Enzyme (Fermentas)
5μl H2O- The digest was carried out in 37°C for 4h.
- The results were visualized on 2% agarose gel.
Results:
- Gel (from left):
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- Sample 1
- Sample 2
- Sample 3
- Sample 4
- Sample 5
- Sample 6
- Sample 7
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- Sample 8
- Sample 9
- Sample 10
- Sample 11
- Sample 12
- Sample 13
- Sample 14
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- Sample 15
- Sample 16
- Sample 17
- Sample 18
- Sample 19
- Sample 20
- Sample 21
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
Conclusions:
- It all looks promising, samples 1, 2, 3, and 7 were sent to be sequenced.
[UPDATE:] The nucleotides in sample 7 read: "tacctctggcggtgata" (prefix and suffix omitted), which means that we have a proper cro-box!.
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