Melbourne/Lab Notebook Weeks 13-16

From 2007.igem.org

< Melbourne(Difference between revisions)

Latest revision as of 05:08, 25 October 2007

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 kb+ ladder
ComA (X/P)
ComA to P4 8J = RA1(X/P) (confirmed)
ComA to P4 8J = RA2(X/P)
ComA to P4 8J = RA3(X/P)
ComA to P4 8J = RA4(X/P)
ComP (X/P)
ComP to P4 8J = RP1 (X/P)(confirmed)
ComP to P4 8J = RP2 (X/P)
ComP to P4 8J = RP3 (X/P)
ComP to P4 8J = RP4 (X/P)
ComP to P3 20I= DP1 (X/P)
ComP to P3 20I= DP2 (X/P)
ComP to P3 20I= DP3 (X/P)
ComP to P3 20I= DP4 (X/P)
1 kb+ ladder

Gel 2:

1 kb+ ladder
ComA to P4 8J = RA1(E/S) -> gel purified 2.2kb fragment
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 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

11 Oct 2007

12 Oct 2007

13 Oct 2007

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Melbourne/Lab Notebook Weeks 13-16

From 2007.igem.org

Latest revision as of 05:08, 25 October 2007

Contents

Week 13

Week 14

Week 15

Week 16

@@ Line 134: / Line 134: @@
 </font><BR>
+*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.
 <font size=3><b>22 Sept 2007
@@ Line 139: / Line 140: @@
 </font><BR>
-*[[Melbourne/Transformation Protocol|Transformed]] into XL-10 gold cells using Quickchange Protocol the ligations from 19/9.
+*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9).
+*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.
 ==Week 14==
@@ Line 150: / Line 152: @@
 </b>
 </font><BR>
+*No Growth observed from cultures BU ABCD of 22/9.
+*[[Melbourne/Miniprep protocol|Miniprepped]]:
+*# Gen-RBS-ComP-ter
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter
+*# [[Melbourne/BBa_J61035|P4 8J]] 1 (22/9)
+*# [[Melbourne/BBa_J61035|P4 8J]] 2 (22/9)
+*# PJS34 1 (22/9)
+*# PJS34 2 (22/9)
+*[[Melbourne/Diagnostic Digest| Digested]]
+*# Gen-RBS-ComP X/P (19/9)
+*# Gen-RBS-ComP-ter X/P
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P (19/9)
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (19/9)
+*# PJS34 X/P
+*# Gen-RBS-ComA E/S
+*# L3 E/X
+[[Melbourne/Loading a DNA gel|Ran on a Gel]] for diagnosing:
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*# Gen-RBS-ComP X/P
+*# Gen-RBS-ComP-ter X/P A
+*# Gen-RBS-ComP-ter X/P B
+*# Gen-RBS-ComP-ter X/P C
+*# Gen-RBS-ComP-ter X/P D
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] X/P
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P A
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P B
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P C
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]]-ter X/P D
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S A
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S B
+*# PJS34 X/P A
+*# PJS34 X/P B
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+[[Melbourne/Loading a DNA gel|Ran on a Gel]] for gel purification:
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*#
+*# Gen-RBS-ComP-ter X/P A <font color=green> band at 700bp </font> (expected 4.6kb band)
+*#
+*# Gen-RBS-ComP-ter X/P D <font color=green> band at 700bp </font>
+*#
+*# Gen-RBS-ComA 3 E/S <font color=green> 2.2kb band taken. </font>
+*#
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)
+*#
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P 740 (5.9)  <font color=green> took 3kb band </font>
+*#
+*# L3 E/X took 2kb band
+*#
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*[[Melbourne/Ligation Protocol|Ligated]] using 10min method:
+*# TetR to RBS-ComP-Ter
+*# Gen-RBS-ComA to Ter
+*# OmpR-c1 to L3
+*# TetR to RBS-[[Melbourne/BBa_I15008|P2 21A]]
+*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] were create for:
+*# PJS34 1 + 2
+*# Gen-RBS-ComP-Ter A <font color=green> not sure </font>
+*# Gen-RBS-[[Melbourne/BBa_I15009|P2 21C]] A <font color=green> not sure </font>
+*[[Melbourne/Transformation Protocol|Transformed]] ligations and plated on Amp plates.
@@ Line 155: / Line 230: @@
 </b>
 </font><BR>
+*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.
@@ Line 160: / Line 237: @@
 </b>
 </font><BR>
-*Miniprepped the following
-**TetR-RBS-P2,21A-ter A,B,C and D
+*[[Melbourne/Miniprep protocol|Miniprepped]] the following
+**TetR-RBS-[[Melbourne/BBa_I15008|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
+**Gen-RBS-ComA-ter A,B,C and D
-**cI-L3 A,B,C and D
+**OmpR-cI-L3 A,B,C and D
+*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] (all were X/P digests):
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 1
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 2
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] (19/9) 3
+*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]]
+*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] A
+*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] B
+*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] C
+*# TetR-RBS-[[Melbourne/BBa_I15008|P2 21A]] D
+*#
+*#
+*#
+*#
+*#
+*# Gen-RBS-ComA-ter D
+*# Gen-RBS-ComA-ter C
+*# Gen-RBS-ComA-ter B
+*# Gen-RBS-ComA-ter A
+*# Gen-RBS-ComA
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*# Omp-c1-L3 A
+*# Omp-c1-L3 B
+*# Omp-c1-L3 C
+*# Omp-c1-L3 D
+*# Death-ComP A
+*# RBS-ComP
+*# Gen-RBS-ComP-ter A
+*# Gen-RBS-ComP-ter B
+*# TetR-RBS-ComP-ter A
+*# TetR-RBS-ComP-ter B
+*# TetR-RBS-ComP-ter C
+*# TetR-RBS-ComP-ter D
+*# [[Melbourne/primary DNA marker|DNA ladder]]
 <font size=3><b>27 Sept 2007
@@ Line 179: / Line 294: @@
 </b>
 </font><BR>
 ==Week 15==
@@ Line 191: / Line 304: @@
 </b>
 </font><BR>
+*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]
+Gel 1:
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*# Gen-RBS-ComA-ter X/P  850bp
+*#
+*# ComA X/P  750bp   <font color=green> purified </font>
+*#
+*# Gen-RBS-ComA 2 X/P  760bp
+*#
+*# Gen-RBS- ComA 3 X/P  760bp    <font color=green> purified although very low yield</font>
+*#
+*#Gen-RBS- ComA 2 E/S  2.2kb
+*#
+*# Gen-RBS-ComA 3 E/S  2.2kb    <font color=green> purified </font>
+*#
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+Gel 2:
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*#
+*# ComP X/P 2.5kb    <font color=green> purified </font>
+*#
+*# [[Melbourne/BBa_I15008|P2 21A]] A X/P  726bp
+*#
+*# [[Melbourne/BBa_I15008|P2 21A]] B X/P  726bp    <font color=green> purified </font>
+*#
+*# [[Melbourne/BBa_Q04510|P2 13K]] S/P  5.4kb    <font color=green> purified </font>
+*#
+*# [[Melbourne/BBa_Q04510|P2 13K]] E/S  1kb    <font color=green> purified </font>
+*#
+*# L3 X/P (9/9)  550bp
+*#
+*# [[Melbourne/BBa_P1010_A|P3 20I]] X/P  2.1kb    <font color=green> purified </font>
+*#
+*# [[Melbourne/BBa_J61035|P4 8J]] S/P  3.5kb    <font color=green> purified </font>
+*#
+*# [[Melbourne/primary DNA marker|DNA ladder]]
+*Also purified [[Melbourne/BBa_J61035|P4 8J]] E/S (1.5kb) on another gel.
+*[[Melbourne/Ligation Protocol|Ligated]] using 10minute methodin 20ul and terminated by placing in -20degC.
+*# Gen-RBS-ComA (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)
+*# ComA (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)
+*# ComP (X/P) to [[Melbourne/BBa_J61035|P4 8J]] (S/P)
+*# ComP (X/P) to [[Melbourne/BBa_P1010_A|P3 20I]] (X/P)
+*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.
@@ Line 201: / Line 370: @@
 </b>
 </font><BR>
+*All plates had colonies from 2/10.
+*4 colonies were picked from each plate of 2/10 and [[Melbourne/Growing up cells|Liquid cultured]].
@@ Line 207: / Line 380: @@
 </font><BR>
+*[[Melbourne/Miniprep protocol|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.
 <font size=3><b>5 Oct 2007
@@ Line 216: / Line 390: @@
 </b>
 </font><BR>
 ==Week 16==
@@ Line 227: / Line 400: @@
 </b>
 </font><BR>
+*[[Melbourne/Diagnostic Digest| Digested]] 10uL of minipreps from 4/10 in 20uL and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:
+Gel1:
+# 1 kb+ ladder
+# ComA (X/P)
+# ComA  to [[Melbourne/BBa_J61035|P4 8J]] = RA1(X/P) (confirmed)
+# ComA  to [[Melbourne/BBa_J61035|P4 8J]] = RA2(X/P)
+# ComA  to [[Melbourne/BBa_J61035|P4 8J]] = RA3(X/P)
+# ComA  to [[Melbourne/BBa_J61035|P4 8J]] = RA4(X/P)
+# ComP (X/P)
+# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP1 (X/P)(confirmed)
+# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP2 (X/P)
+# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP3 (X/P)
+# ComP to [[Melbourne/BBa_J61035|P4 8J]] = RP4 (X/P)
+# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP1 (X/P)
+# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP2 (X/P)
+# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP3 (X/P)
+# ComP to [[Melbourne/BBa_P1010_A|P3 20I]]= DP4 (X/P)
+# 1 kb+ ladder
+Gel 2:
+# 1 kb+ ladder
+#
+# ComA  to [[Melbourne/BBa_J61035|P4 8J]] = RA1(E/S) -> gel purified 2.2kb 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.
+*# RA1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GAT
+*# RP1 (gel purified) (E/S) to [[Melbourne/BBa_B0014|P1 1G]] (E/X)= GPT
+*[[Melbourne/Transformation Protocol|Transformed]] 10ul of each ligation and plated on Amp plates.
 <font size=3><b>9 Oct 2007
@@ Line 233: / Line 439: @@
 </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
 </b>
 </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