Team:TU Delft/8 July 2010 content

From 2010.igem.org

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(Lab work)
 
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==Lab work==
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=Lab work=
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<h4>Ordered DNA stocks</h4>
+
==Ordered DNA stocks==
-
We harvested the 200 mL bacterial cells of the [http://2010.igem.org/Team:TU_Delft#/blog?blog=6_July_2010 16 DNA parts]. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]]. With the rest we centrifuged at 4,000 rmp for 15 minutes and stored the pellets in the -20 °C.
+
We harvested the 200 mL bacterial cells of the [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=6_July_2010 16 DNA parts]. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]]. With the rest we centrifuged at 4,000 rmp for 15 minutes and stored the pellets in the -20 °C.
-
<h4>Characterization of Anderson RBS sequences</h4>
+
==Characterization of Anderson RBS sequences==
-
[http://2010.igem.org/Team:TU_Delft#/blog?blog=6_July_2010 Tuesday's] ligation products of BioBricks K398500-K398504 yielded successful transformants. [[Team:TU_Delft/protocols/colony_PCR|Single colony PCRs]] were performed and loaded onto a gel. The bands on the [[Team:TU_Delft/protocols/agarose_gel |1% agarose gel]] indicated the presence of inserts with the proper lengths:
+
[http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=6_July_2010 Tuesday's] ligation products of BioBricks K398500-K398504 yielded successful transformants. [[Team:TU_Delft/protocols/colony_PCR|Single colony PCRs]] were performed and loaded onto a gel. The bands on the [[Team:TU_Delft/protocols/agarose_gel |1% agarose gel]] indicated the presence of inserts with the proper lengths:
-
[[Image:C-PCR_transformants_08-July.png|400px|thumb|left|1% Agarose gel of scPCR]]
+
[[Image:C-PCR_transformants_08-July.png|400px|thumb|left|1 % agarose of colony PCR. Gel runned 1 hour at 100 V. Of all samples 5μL + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
Lane descriptions:
Lane descriptions:
Line 14: Line 14:
|'''Description'''
|'''Description'''
|'''Expected Length (bp)'''
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
|'''Remarks'''
|'''Remarks'''
|-
|-
|1
|1
-
|scPCR product of K398500 transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation mix K398500
-
|
+
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|Unexpected band at 2700
|Unexpected band at 2700
|-
|-
|2
|2
-
|scPCR product of K398500 transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation mix K398500
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|3
|3
-
|scPCR product of K398501 transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation mix K398501
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|4
|4
-
|scPCR product of K398501 transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation mix K398501
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|5
|5
-
|scPCR product of K398502 transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation mix K398502
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|6
|6
-
|scPCR product of K398502 transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation mix K398502
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|7
|7
-
|scPCR product of K398503 transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation mix K398503
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|8
|8
-
|scPCR product of K398503 transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation mix K398503
-
|
+
|1158
-
|Visible
+
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|probably run of the gel
|-
|-
|9
|9
-
|scPCR product of K398504 transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation mix K398504
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
|
|
-
|Visible
 
|-
|-
|10
|10
-
|scPCR product of K398504 transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation mix K398504
-
|
+
|1158
-
|Visible
+
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|Sample not fully loaded on gel
|-
|-
|11
|11
-
|scPCR product of ligase control transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of ligation control
|
|
 +
|G00100 + G00101
|
|
 +
|probably run of the gel
 +
|-
|12
|12
-
|scPCR product of ligase control transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of ligation control
|
|
 +
|G00100 + G00101
|
|
 +
|
 +
|-
|13
|13
-
|scPCR product of digestion control transformant #1 (5 μL + 1 μL loadingbuffer)
+
|transformant #1 of digestion control
|
|
 +
|G00100 + G00101
|
|
 +
|probably run of the gel
 +
|-
|14
|14
-
|scPCR product of digestion control transformant #2 (5 μL + 1 μL loadingbuffer)
+
|transformant #2 of digestion control
|
|
 +
|G00100 + G00101
|
|
-
|15
+
|
 +
|-
 +
|M1
|SmartLadder Marker
|SmartLadder Marker
-
|Varying
+
|n/a
-
|Visible
+
|n/a
 +
|n/a
 +
|
|}
|}
The colonies belonging to lanes 2, 4, 6, 8 and 10 were plated out on AMP plates to yield reincultures for subsequent characterization experiments.
The colonies belonging to lanes 2, 4, 6, 8 and 10 were plated out on AMP plates to yield reincultures for subsequent characterization experiments.
-
<h4>Alkane degradation</h4>
+
==Alkane degradation==
Today we got the Gas Chromatograph working, we could identify several peaks. Now we are ready for the real experiments!
Today we got the Gas Chromatograph working, we could identify several peaks. Now we are ready for the real experiments!
[[Image:Team_TU_Delft_GCtest.PNG|400px|thumb|left]]
[[Image:Team_TU_Delft_GCtest.PNG|400px|thumb|left]]

Latest revision as of 19:40, 5 August 2010

Contents

Lab work

Ordered DNA stocks

We harvested the 200 mL bacterial cells of the 16 DNA parts. We used 3 mL of the bacterial cells to make -80 °C stocks. With the rest we centrifuged at 4,000 rmp for 15 minutes and stored the pellets in the -20 °C.

Characterization of Anderson RBS sequences

Tuesday's ligation products of BioBricks K398500-K398504 yielded successful transformants. Single colony PCRs were performed and loaded onto a gel. The bands on the 1% agarose gel indicated the presence of inserts with the proper lengths:

1 % agarose of colony PCR. Gel runned 1 hour at 100 V. Of all samples 5μL + 1 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane descriptions:

# Description Expected Length (bp) Primers Status Remarks
1 transformant #1 of ligation mix K398500 1158 G00100 + G00101 Unexpected band at 2700
2 transformant #2 of ligation mix K398500 1158 G00100 + G00101
3 transformant #1 of ligation mix K398501 1158 G00100 + G00101
4 transformant #2 of ligation mix K398501 1158 G00100 + G00101
5 transformant #1 of ligation mix K398502 1158 G00100 + G00101
6 transformant #2 of ligation mix K398502 1158 G00100 + G00101
7 transformant #1 of ligation mix K398503 1158 G00100 + G00101
8 transformant #2 of ligation mix K398503 1158 G00100 + G00101 probably run of the gel
9 transformant #1 of ligation mix K398504 1158 G00100 + G00101
10 transformant #2 of ligation mix K398504 1158 G00100 + G00101 Sample not fully loaded on gel
11 transformant #1 of ligation control G00100 + G00101 probably run of the gel
12 transformant #2 of ligation control G00100 + G00101
13 transformant #1 of digestion control G00100 + G00101 probably run of the gel
14 transformant #2 of digestion control G00100 + G00101
M1 SmartLadder Marker n/a n/a n/a

The colonies belonging to lanes 2, 4, 6, 8 and 10 were plated out on AMP plates to yield reincultures for subsequent characterization experiments.

Alkane degradation

Today we got the Gas Chromatograph working, we could identify several peaks. Now we are ready for the real experiments!

Team TU Delft GCtest.PNG