Preparation of biobricks to send

Marcin

Task 1: Preparation of bacterial cultures:

Comment:

I prepare liquid bacterial cultures for isolation of followed biobricks:

• BBa_K177016
• BBa_K177035
• BBa_K177036
• BBa_K177042
• BBa_K177044
• BBa_K177046

Task 2: Isolation of plasmid from cultures described in Task 1

Methods:

• Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Assembly of endosome detection operon

Marcin

Task 1: DNA digest to create following biobricks:

• BBa_K177037
• BBa_K177044

Methods:

• Constructs to digest:
  • BBa_K177035 - PstI, SpeI
  • BBa_K177035 - PstI, XbaI
  • BBa_K177036 - PstI, SpeI
  • BBa_K177044 - PstI, XbaI
• Reaction mixture composition:

15 μl purified plasmid DNA product
1 μl enzyme 1 (Fermentas)
1 μl enzyme 2 (Fermentas) 
5 μl Buffer Tango (Fermentas)
28 μl MQ water

• Reactions were carried out 9 hour

Cloning of the mgtc promoter 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 1% 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. Reference sample
8. Sample 6
9. Sample 7
10. Sample 8
11. Sample 9
12. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)

Conclusions:

• No hits, just a lot of "junk".

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. Reference sample
8. Sample 6
9. Sample 7
10. Sample 8
11. Sample 9
12. Sample 10

Conclusions:

• No hits, all plasmids empty.