Isolation of BBa_J63010 from 2009 Kit

Monika

Task:

• Isolate BioBrick [http://partsregistry.org/Part:BBa_J63010 BBa_J63010] from 2009 Kit Plate 1

Methods:

• Alkaline lysis with A&A Biotechnology Kit, procedure described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf 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):
1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. Sample 1
3. Sample 2
4. Sample 3
5. Sample 4
6. Sample 5
7. Sample 6
8. Sample 7
9. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
10. Sample 8
11. Sample 9
12. Sample 10
13. Sample 11
14. Sample 12
15. Sample 13
16. Sample 14
17. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
18. Sample 15
19. Sample 16
20. Sample 17
21. Sample 18
22. Sample 19
23. Sample 20
24. Sample 21
25. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)

Conclusions:

• It all looks promising, samples 1, 2, 3, and 7 were sent to be sequenced.