Team:TU Delft/8 July 2010 content

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===Lab work===
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=Lab work=
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Today we got the Gas Chromatograph working, we could identify several peaks. Now we are ready for the real experiments!
+
==Ordered DNA stocks==
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[[Image:Team_TU_Delft_GCtest.PNG|300px|thumb|left]]
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We harvested the 200 mL bacterial cells of the [https://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.
 +
==Characterization of Anderson RBS sequences==
 +
[https://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:
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We harvested the 200 mL bacterial cells of the [https://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.
+
[[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]]
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====Characterization of Anderson RBS sequences====
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Lane descriptions:
 +
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
 +
|'''Description'''
 +
|'''Expected Length (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|'''Remarks'''
 +
|-
 +
|1
 +
|transformant #1 of ligation mix K398500
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|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|Unexpected band at 2700
 +
|-
 +
|2
 +
|transformant #2 of ligation mix K398500
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|3
 +
|transformant #1 of ligation mix K398501
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|4
 +
|transformant #2 of ligation mix K398501
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|5
 +
|transformant #1 of ligation mix K398502
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|6
 +
|transformant #2 of ligation mix K398502
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|7
 +
|transformant #1 of ligation mix K398503
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|8
 +
|transformant #2 of ligation mix K398503
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|probably run of the gel
 +
|-
 +
|9
 +
|transformant #1 of ligation mix K398504
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|
 +
|-
 +
|10
 +
|transformant #2 of ligation mix K398504
 +
|1158
 +
|G00100 + G00101
 +
|<font color=limegreen>✓</font>
 +
|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
 +
|
 +
|
 +
|-
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|M1
 +
|SmartLadder Marker
 +
|n/a
 +
|n/a
 +
|n/a
 +
|
 +
|}
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Yesterday's ligation products containing I13401 and the 5 Anderson RBS plasmids (J61100, J61101, J51107, J61117, J61127) yielded successful transformants! The single colony PCRs performed indicated the presence of inserts with the proper lengths. These colonies were further plated out to yield reincultures for subsequent characterization.
+
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!
 +
[[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
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