Melbourne/Lab Notebook Weeks 13-16

From 2007.igem.org

(Difference between revisions)
(→Week 14)
(→Week 14)
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*# PJS34 2 (22/9)
*# PJS34 2 (22/9)
-
*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]]:
+
*[[Melbourne/Diagnostic Digest| Digested]]
*# Gen-RBS-ComP X/P (19/9)
*# Gen-RBS-ComP X/P (19/9)
*# Gen-RBS-ComP-ter X/P
*# Gen-RBS-ComP-ter X/P
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*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] E/S (19/9)
*# Gen-RBS-[[Melbourne/BBa_I15008|P2 21A]] X/P (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.
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</b>
</b>
</font><BR>
</font><BR>
 +
 +
*[[Melbourne/Growing up cells|Liquid cultured]] transformations from 24/9.
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</b>
</b>
</font><BR>
</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
**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]]
 +
 
 +
 
<font size=3><b>27 Sept 2007  
<font size=3><b>27 Sept 2007  

Revision as of 13:45, 9 October 2007

<Return to Lab notebook> <team home page>

Contents

Week 13

16 Sept 2007


17 Sept 2007

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.


18 Sept 2007

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


19 Sept 2007

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


    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

Week 14

23 Sept 2007


24 Sept 2007

  • No Growth observed from cultures BU ABCD of 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



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

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