Team:TU Delft/16 July 2010 content

From 2010.igem.org

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(Characterization of Anderson RBS sequences)
 
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=Lab work=
=Lab work=
-
<h4>Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing</h4>
+
==Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing==
-
The colony PCR of [http://2010.igem.org/Team:TU_Delft#/blog?blog=15_July_2010 yesterday] was put on[[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]   
+
The colony PCR of [http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=15_July_2010 yesterday] was put on[[Team:TU_Delft/protocols/agarose_gel|1% agarose gel]]   
 +
 
 +
[[Image:TU Delft E 20100716 PCR.jpg|450px|1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker]]
 +
 
 +
Lane description:
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''Description'''
 +
|'''Expected lenght (bp)'''
 +
|'''Primers'''
 +
|'''Status'''
 +
|-
 +
|0
 +
|SmartLadder
 +
|n/a
 +
|n/a
 +
|n/a
 +
|-
 +
|1
 +
|transformant #1 of ligation mix AlkS + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|2
 +
|transformant #2 of ligation mix AlkS + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|3
 +
|transformant #3 of ligation mix AlkS + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|4
 +
|transformant #4 of ligation mix AlkS + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|5
 +
|transformant #1 of ligation mix ladA + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|6
 +
|transformant #2 of ligation mix ladA + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|7
 +
|transformant #1 of ligation mix RubA3 + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|8
 +
|transformant #2 of ligation mix RubA3 + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|9
 +
|transformant #3 of ligation mix RubA3 + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|10
 +
|transformant #4 of ligation mix RubA3 + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|11
 +
|transformant #5 of ligation mix RubA3 + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|12
 +
|transformant #1 of ligation mix PhPFDbeta + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|13
 +
|transformant #1 of ligation mix AlnA + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|14
 +
|transformant #2 of ligation mix AlnA + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|15
 +
|transformant #1 of ligation mix OprG + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|16
 +
|transformant #2 of ligation mix OprG + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|17
 +
|transformant #3 of ligation mix OprG + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|18
 +
|transformant #1 of ligation mix PhPFDalpha + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|19
 +
|transformant #2 of ligation mix PhPFDalpha + pSB1C3
 +
|
 +
|G00100 + G00101
 +
|<font color=red>✗</font>
 +
|-
 +
|}
 +
 
 +
 
We harvested the 1 mL bacterial cells of possible correct transformations. The pellets were stored at -20 °C.
We harvested the 1 mL bacterial cells of possible correct transformations. The pellets were stored at -20 °C.
Line 8: Line 142:
==Characterization of Anderson RBS sequences==
==Characterization of Anderson RBS sequences==
-
<h5>Fluorescence measurements Attempt #2</h5>
+
====Fluorescence measurements Attempt #2====
 +
 
Data analysis to follow shortly (good stuff!)
Data analysis to follow shortly (good stuff!)
-
<h5>Assembly of reference construct</h5>
+
====Assembly of reference construct & positive control====
-
The [[Team:TU_Delft/protocols/birnboim_plasmid_isolation Birnboim method ]] was used to isolate J23100 and I13522 (both in pSB1A2) from the o/n 5 mL LB cultures.
+
 
 +
[http://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=15_July_2010 Yesterday's] digestion products of K081005 and I13401 were set for ligation over the weekend at 4 °C:
 +
 
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''#'''
 +
|'''BioBrick'''
 +
|'''Fragment'''
 +
|'''Recipient plasmid'''
 +
|'''Final volume'''
 +
|-
 +
|1
 +
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
 +
|17.5 μL ‘E-K081005-S’
 +
|4 μL ‘E-pSB1A2 I13401-X’
 +
|25 μL
 +
|-
 +
|2
 +
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
 +
|17.5 μL ‘E-K081005-S’
 +
|4 μL ‘E-pSB1A2 I13401-X
 +
|25 μL
 +
|-
 +
|3
 +
|Ligation control
 +
|None
 +
|4 μL ‘E-pSB1A2 I13401-X
 +
|25 μL
 +
|}
 +
 
 +
To all samples with an end volume of 25 μL, 2.5 μL Ligase buffer was added.
 +
 
 +
The [[Team:TU_Delft/protocols/birnboim_plasmid_isolation|Birnboim method]] was used to isolate J23100 and I13522 (both in pSB1A2) from the o/n 5 mL LB cultures. We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]] . The following concentrations of plasmid were obtained:
 +
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''BioBrick'''
 +
|'''Concentration (ng/μL)'''
 +
|-
 +
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 J23100]
 +
|130.7
 +
|-
 +
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 I13522]
 +
|106.7
 +
|}

Latest revision as of 20:22, 3 August 2010

Contents

Lab work

Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing

The colony PCR of yesterday was put on1% agarose gel

1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 μL + 2 μL loadingbuffer was loaded. 5 μL was loaded of marker

Lane description:

# Description Expected lenght (bp) Primers Status
0 SmartLadder n/a n/a n/a
1 transformant #1 of ligation mix AlkS + pSB1C3 G00100 + G00101
2 transformant #2 of ligation mix AlkS + pSB1C3 G00100 + G00101
3 transformant #3 of ligation mix AlkS + pSB1C3 G00100 + G00101
4 transformant #4 of ligation mix AlkS + pSB1C3 G00100 + G00101
5 transformant #1 of ligation mix ladA + pSB1C3 G00100 + G00101
6 transformant #2 of ligation mix ladA + pSB1C3 G00100 + G00101
7 transformant #1 of ligation mix RubA3 + pSB1C3 G00100 + G00101
8 transformant #2 of ligation mix RubA3 + pSB1C3 G00100 + G00101
9 transformant #3 of ligation mix RubA3 + pSB1C3 G00100 + G00101
10 transformant #4 of ligation mix RubA3 + pSB1C3 G00100 + G00101
11 transformant #5 of ligation mix RubA3 + pSB1C3 G00100 + G00101
12 transformant #1 of ligation mix PhPFDbeta + pSB1C3 G00100 + G00101
13 transformant #1 of ligation mix AlnA + pSB1C3 G00100 + G00101
14 transformant #2 of ligation mix AlnA + pSB1C3 G00100 + G00101
15 transformant #1 of ligation mix OprG + pSB1C3 G00100 + G00101
16 transformant #2 of ligation mix OprG + pSB1C3 G00100 + G00101
17 transformant #3 of ligation mix OprG + pSB1C3 G00100 + G00101
18 transformant #1 of ligation mix PhPFDalpha + pSB1C3 G00100 + G00101
19 transformant #2 of ligation mix PhPFDalpha + pSB1C3 G00100 + G00101


We harvested the 1 mL bacterial cells of possible correct transformations. The pellets were stored at -20 °C. We used 3 mL of the bacterial cells to make -80 °C stocks.

Characterization of Anderson RBS sequences

Fluorescence measurements Attempt #2

Data analysis to follow shortly (good stuff!)

Assembly of reference construct & positive control

Yesterday's digestion products of K081005 and I13401 were set for ligation over the weekend at 4 °C:

# BioBrick Fragment Recipient plasmid Final volume
1 K136011 17.5 μL ‘E-K081005-S’ 4 μL ‘E-pSB1A2 I13401-X’ 25 μL
2 K136011 17.5 μL ‘E-K081005-S’ 4 μL ‘E-pSB1A2 I13401-X 25 μL
3 Ligation control None 4 μL ‘E-pSB1A2 I13401-X 25 μL

To all samples with an end volume of 25 μL, 2.5 μL Ligase buffer was added.

The Birnboim method was used to isolate J23100 and I13522 (both in pSB1A2) from the o/n 5 mL LB cultures. We used 3 mL of the bacterial cells to make -80 °C stocks . The following concentrations of plasmid were obtained:

BioBrick Concentration (ng/μL)
J23100 130.7
I13522 106.7