Melbourne/Lab Notebook Weeks 13-16

From 2007.igem.org

< Melbourne(Difference between revisions)
(→Week 14)
(→Week 16)
 
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*[[Melbourne/Growing up cells|Liquid cultured]] 4 colonies from each of cultures (21/9).  
*[[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 JS34 transformation.
+
*Also [[Melbourne/Growing up cells|Liquid cultured]] 2 colonies in Amp from [[Melbourne/Lab BL Notebook/PsrfA sequence|PJS34]] transformation.
==Week 14==
==Week 14==
Line 163: Line 163:
*# 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
Line 170: Line 170:
*# 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]] (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  
<font size=3><b>27 Sept 2007  
Line 210: Line 304:
</b>
</b>
</font><BR>
</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.
 +
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</b>
</b>
</font><BR>
</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]].
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</font><BR>
</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  
<font size=3><b>5 Oct 2007  
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</b>
</b>
</font><BR>
</font><BR>
-
 
==Week 16==
==Week 16==
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</b>
</b>
</font><BR>
</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  
<font size=3><b>9 Oct 2007  
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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

<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

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:


  • 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

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

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

Retrieved from "http://2007.igem.org/wiki/index.php/Melbourne/Lab_Notebook_Weeks_13-16"