Melbourne/Lab Notebook Weeks 13-16

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Revision as of 13:11, 9 October 2007

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Week 13

16 Sept 2007

17 Sept 2007

• Miniprepped 2 cultures from each of the transformations on 12/9 and 4 cultures from Omp-C1-reporter.
• Digested these and Ran on a Gel:

Gel1:

• 1. DNA ladder
  2. P17OA E/H
  3. P17OB E/H
  4. P1 5G A E/H
  5. P1 5G B E/H
  6. Omp-C1-reporter A X/P
  7. Omp-C1-reporter B X/P
  8. Omp-C1-reporter C X/P
  9. Omp-C1-reporter D X/P
  10. P2 21A A X/P
  11. P2 21A B X/P
  12. P2 21C A X/P
  13. P2 21C B X/P
  14. Death-ComA 5min ligation A X/P
  15. Death-ComA 5min ligation B X/P
  16. Death-ComA overnight ligation A X/P
  17. Death-ComA overnight ligation B X/P
  18. DNA ladder

Gel2:

• 1. DNA ladder
  2. Death-ComP A X/P
  3. Death-ComP B X/P
  4. Cph8 A X/P
  5. Cph8 B X/P

-All had desired insert except for lanes 8-11 (OmpR-C1-reporter) in Gel 1 and lanes 4, 5 (CpH8) in Gel 2.

• Gel purified:
  1. P2 21A E/X
  2. P2 21C E/X
  3. Death-CompA E/X
  4. Death-ComP E/X
  5. P4 8J A E/S
  6. P4 8J B E/S

• Ligated using 1hour ligation method at room temperature:
  • P4 8J E/S + P2 21A E/X
  • P4 8J E/S + P2 21C E/X
  • P4 8J E/S + Death-CompA E/X
  • P4 8J E/S + Death-ComP E/X

• Transformed above and plated.

18 Sept 2007

• Liquid cultured 4 colonies each from transformants of 17/9 (2 from Death-ComA).

19 Sept 2007

• Miniprepped all cultures from 18/9.
• Digested and Ran on a Gel:

• 1. DNA ladder
  2. Gen-RBS-ComP 1 E/S
  3. Gen-RBS-ComP 2 E/S
  4. Gen-RBS-ComP 3 E/S
  5. Gen-RBS-ComP 4 E/S
  6. Gen-RBS-P2 21A 1 X/P
  7. Gen-RBS-P2 21A 2 X/P
  8. Gen-RBS-P2 21A 3 X/P
  9. Gen-RBS-P2 21A 4 X/P
  10. Gen-RBS-P2 21C 1 X/S
  11. Gen-RBS-P2 21C 2 X/S
  12. Gen-RBS-P2 21C 3 X/S
  13. Gen-RBS-P2 21C 4 X/S
  14. Gen-RBS-ComA 1 E/S
  15. Gen-RBS-ComA 2 E/S
  16. DNA ladder

-Ran at 100V for 45min. -Lanes 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, and 15 had desired bands.

• Digested and Ran on a Gel for gel purification:
  1. DNA ladder
  3. Gen-RBS-ComP E/S 3.9kb
  5. Gen-RBS-ComA E/S 2.1kb Had no DNA
  7. Gen-RBS-P2 21A X/P 2.2kb Took wrong fragment
  9. Gen-RBS-P2 21C E/S 2.2kb
  11. L3 X/P 550bp Ran off
  13. P17O S/P 2kb
  16. P1 5G E/X 3.3kb
  17. P1 5G E/X 3.3kb
  18. P1 5G E/X 3.3kb
  20. DNA ladder

• Ligated with 5min method:

• 1. Gen-RBS-ComP E/S into P1 5G E/X -survives on G/A
  2. Gen-RBS-P2 21C E/S into P1 5G E/X -survives on G/K

20 Sept 2007

21 Sept 2007

22 Sept 2007

• Transformed into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.

Week 14

23 Sept 2007

24 Sept 2007

25 Sept 2007

26 Sept 2007

• Miniprepped the following
  • TetR-RBS-P2,21A-ter A,B,C and D
  • TetR-RBS-ComP-ter A,B,C and D
  • GenRBS-ComA-ter A,B,C and D
  • cI-L3 A,B,C and D

27 Sept 2007

28 Sept 2007

29 Sept 2007

Week 15

30 Sept 2007

1 Oct 2007

2 Oct 2007

3 Oct 2007

4 Oct 2007

5 Oct 2007

6 Oct 2007

Week 16

7 Oct 2007

8 Oct 2007

9 Oct 2007

10 Oct 2007

11 Oct 2007

12 Oct 2007

13 Oct 2007