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	# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment		# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (E/S) -> gel purified 4kb fragment
	#		#
		+
	*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.		*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.
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	</font><BR>		</font><BR>
		+	*All plates had colonies from 8/10.
		+	*4 colonies were picked from each plate of 8/10 and [[Melbourne/Growing up cells|Liquid cultured]].
	<font size=3><b>10 Oct 2007		<font size=3><b>10 Oct 2007
	</b>		</b>
	</font><BR>		</font><BR>
		+
		+	*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 9/10.
		+	*[[Melbourne/Diagnostic Digest| Digested]]  minipreps  in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:
		+
		+	Gel1:
		+	# 1 kb+ ladder
		+	# ComA (X/P)
		+	# RA1(X/P)
		+	# GAT1 (X/P)
		+	# GAT2 (X/P)(confirmed)
		+	# GAT3 (X/P)
		+	# GAT4 (X/P)
		+	# ComP (X/P)
		+	# RP1 (X/P)
		+	# GPT1 (X/P)
		+	# GPT2 (X/P)
		+	# GPT3 (X/P)
		+	# GPT4 (X/P)(confirmed)
		+	# 1 kb+ ladder
		+
		+	GAT2= GenRBS-ComA-ter
		+	GPT4= GenRBS-ComP-ter -> need sequence confirmation

---

Latest revision as of 05:08, 25 October 2007

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Contents 1 Week 13 2 Week 14 3 Week 15 4 Week 16

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

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

22 Sept 2007

• Liquid cultured 4 colonies from each of cultures (21/9).
• Also Liquid cultured 2 colonies in Amp from PJS34 transformation.

Week 14

23 Sept 2007

24 Sept 2007

• No Growth observed from cultures BU ABCD of 22/9.

• Miniprepped:
  1. Gen-RBS-ComP-ter
  2. Gen-RBS-P2 21C-ter
  3. P4 8J 1 (22/9)
  4. P4 8J 2 (22/9)
  5. PJS34 1 (22/9)
  6. PJS34 2 (22/9)

• Digested
  1. Gen-RBS-ComP X/P (19/9)
  2. Gen-RBS-ComP-ter X/P
  3. Gen-RBS-P2 21C-ter X/P
  4. Gen-RBS-P2 21C X/P (19/9)
  5. Gen-RBS-P2 21A E/S (19/9)
  6. Gen-RBS-P2 21A X/P (19/9)
  7. PJS34 X/P
  8. Gen-RBS-ComA E/S
  9. L3 E/X

Ran on a Gel for diagnosing:

• 1. DNA ladder
  2. Gen-RBS-ComP X/P
  3. Gen-RBS-ComP-ter X/P A
  4. Gen-RBS-ComP-ter X/P B
  5. Gen-RBS-ComP-ter X/P C
  6. Gen-RBS-ComP-ter X/P D
  7. Gen-RBS-P2 21C X/P
  8. Gen-RBS-P2 21C-ter X/P A
  9. Gen-RBS-P2 21C-ter X/P B
  10. Gen-RBS-P2 21C-ter X/P C
  11. Gen-RBS-P2 21C-ter X/P D
  12. Gen-RBS-P2 21A E/S A
  13. Gen-RBS-P2 21A E/S B
  14. PJS34 X/P A
  15. PJS34 X/P B
  16. DNA ladder

Ran on a Gel for gel purification:

• 1. DNA ladder
  3. Gen-RBS-ComP-ter X/P A band at 700bp (expected 4.6kb band)
  5. Gen-RBS-ComP-ter X/P D band at 700bp
  7. Gen-RBS-ComA 3 E/S 2.2kb band taken.
  9. Gen-RBS-P2 21A X/P 740 (5.9)
  11. Gen-RBS-P2 21A X/P 740 (5.9) took 3kb band
  13. L3 E/X took 2kb band
  15. DNA ladder

• Ligated using 10min method:
  1. TetR to RBS-ComP-Ter
  2. Gen-RBS-ComA to Ter
  3. OmpR-c1 to L3
  4. TetR to RBS-P2 21A

• Glycerol Stocks were create for:
  1. PJS34 1 + 2
  2. Gen-RBS-ComP-Ter A not sure
  3. Gen-RBS-P2 21C A not sure

• Transformed ligations and plated on Amp plates.

25 Sept 2007

• Liquid cultured transformations from 24/9.

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
  • Gen-RBS-ComA-ter A,B,C and D
  • OmpR-cI-L3 A,B,C and D

• Digested and Ran on a Gel (all were X/P digests):
  1. DNA ladder
  2. Gen-RBS-P2 21A (19/9) 1
  3. Gen-RBS-P2 21A (19/9) 2
  4. Gen-RBS-P2 21A (19/9) 3
  5. Gen-RBS-P2 21A
  6. TetR-RBS-P2 21A A
  7. TetR-RBS-P2 21A B
  8. TetR-RBS-P2 21A C
  9. TetR-RBS-P2 21A D
  15. Gen-RBS-ComA-ter D
  16. Gen-RBS-ComA-ter C
  17. Gen-RBS-ComA-ter B
  18. Gen-RBS-ComA-ter A
  19. Gen-RBS-ComA
  20. DNA ladder
  21. DNA ladder
  22. Omp-c1-L3 A
  23. Omp-c1-L3 B
  24. Omp-c1-L3 C
  25. Omp-c1-L3 D
  26. Death-ComP A
  27. RBS-ComP
  28. Gen-RBS-ComP-ter A
  29. Gen-RBS-ComP-ter B
  30. TetR-RBS-ComP-ter A
  31. TetR-RBS-ComP-ter B
  32. TetR-RBS-ComP-ter C
  33. TetR-RBS-ComP-ter D
  34. DNA ladder

27 Sept 2007

28 Sept 2007

29 Sept 2007

Week 15

30 Sept 2007

1 Oct 2007

• Digested and Ran on a Gel

Gel 1:

• 1. DNA ladder
  2. Gen-RBS-ComA-ter X/P 850bp
  4. ComA X/P 750bp purified
  6. Gen-RBS-ComA 2 X/P 760bp
  8. Gen-RBS- ComA 3 X/P 760bp purified although very low yield
  10. Gen-RBS- ComA 2 E/S 2.2kb
  12. Gen-RBS-ComA 3 E/S 2.2kb purified
  14. DNA ladder

Gel 2:

• 1. DNA ladder
  3. ComP X/P 2.5kb purified
  5. P2 21A A X/P 726bp
  7. P2 21A B X/P 726bp purified
  9. P2 13K S/P 5.4kb purified
  11. P2 13K E/S 1kb purified
  13. L3 X/P (9/9) 550bp
  15. P3 20I X/P 2.1kb purified
  17. P4 8J S/P 3.5kb purified
  19. DNA ladder

• Also purified P4 8J E/S (1.5kb) on another gel.

• Ligated using 10minute methodin 20ul and terminated by placing in -20degC.
  1. Gen-RBS-ComA (E/S) to P1 1G (E/X)
  2. ComA (X/P) to P4 8J (S/P)
  3. ComP (X/P) to P4 8J (S/P)
  4. ComP (X/P) to P3 20I (X/P)

• Transformed 10ul of each ligation and plated on Amp plates.

2 Oct 2007

3 Oct 2007

• All plates had colonies from 2/10.
• 4 colonies were picked from each plate of 2/10 and Liquid cultured.

4 Oct 2007

• Miniprepped cultures from 3/10. Added 250ul of Nuclease free-water/TE buffer instead of 50ul in the final elution step. The DNA may be too dilute.

5 Oct 2007

6 Oct 2007

Week 16

7 Oct 2007

8 Oct 2007

• Digested 10uL of minipreps from 4/10 in 20uL and Ran on a Gel:

Gel1:

1. 1 kb+ ladder
2. ComA (X/P)
3. ComA to P4 8J = RA1(X/P) (confirmed)
4. ComA to P4 8J = RA2(X/P)
5. ComA to P4 8J = RA3(X/P)
6. ComA to P4 8J = RA4(X/P)
7. ComP (X/P)
8. ComP to P4 8J = RP1 (X/P)(confirmed)
9. ComP to P4 8J = RP2 (X/P)
10. ComP to P4 8J = RP3 (X/P)
11. ComP to P4 8J = RP4 (X/P)
12. ComP to P3 20I= DP1 (X/P)
13. ComP to P3 20I= DP2 (X/P)
14. ComP to P3 20I= DP3 (X/P)
15. ComP to P3 20I= DP4 (X/P)
16. 1 kb+ ladder

Gel 2:

1. 1 kb+ ladder
3. ComA to P4 8J = RA1(E/S) -> gel purified 2.2kb fragment
5. ComP to P4 8J = RP1 (E/S) -> gel purified 4kb fragment

• Ligated using 10minute methodin 20ul and terminated by placing in -20degC.
  1. RA1 (gel purified) (E/S) to P1 1G (E/X)= GAT
  2. RP1 (gel purified) (E/S) to P1 1G (E/X)= GPT

• Transformed 10ul of each ligation and plated on Amp plates.

9 Oct 2007

• All plates had colonies from 8/10.
• 4 colonies were picked from each plate of 8/10 and Liquid cultured.

10 Oct 2007

• Miniprepped cultures from 9/10.
• Digested minipreps in 20uL and Ran on a Gel:

Gel1:

1. 1 kb+ ladder
2. ComA (X/P)
3. RA1(X/P)
4. GAT1 (X/P)
5. GAT2 (X/P)(confirmed)
6. GAT3 (X/P)
7. GAT4 (X/P)
8. ComP (X/P)
9. RP1 (X/P)
10. GPT1 (X/P)
11. GPT2 (X/P)
12. GPT3 (X/P)
13. GPT4 (X/P)(confirmed)
14. 1 kb+ ladder

GAT2= GenRBS-ComA-ter GPT4= GenRBS-ComP-ter -> need sequence confirmation

11 Oct 2007

12 Oct 2007

13 Oct 2007