Team:TU Delft/16 July 2010 content

From 2010.igem.org

(Difference between revisions)
(→Characterization of Anderson RBS sequences)
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We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
We used 3 mL of the bacterial cells to make [[Team:TU_Delft/protocols/freezing_bacterial_stocks|-80 °C stocks]].
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==Characterization of Anderson RBS sequences==
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<h4>Characterization of Anderson RBS sequences</h4>  
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<h5>Fluorescence measurements Attempt #2</h5>
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'''Fluorescence measurements Attempt #2'''
 +
 
Data analysis to follow shortly (good stuff!)
Data analysis to follow shortly (good stuff!)
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<h5>Assembly of reference construct & positive control</h5>
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'''Assembly of reference construct & positive control'''
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[https://2010.igem.org/Team:TU_Delft#/blog?blog=15_July_2010 Yesterday's] digestion products of K081005 and I13401 were set for ligation over the weekend:
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 +
[https://2010.igem.org/Team:TU_Delft#/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"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''#'''
|'''#'''
|'''BioBrick'''
|'''BioBrick'''
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|'''Insert fragment'''
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|'''Fragment'''
|'''Recipient plasmid'''
|'''Recipient plasmid'''
|'''Final volume'''
|'''Final volume'''
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|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
|17.5 μL ‘E-K081005-S’
|17.5 μL ‘E-K081005-S’
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|4 μL ‘E-pSB1A2-I13401-X’
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|4 μL ‘E-pSB1A2 I13401-X’
|25 μL
|25 μL
|-
|-
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|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K136011 K136011]
|17.5 μL ‘E-K081005-S’
|17.5 μL ‘E-K081005-S’
-
|4 μL ‘E-pSB1A2-I13401-X
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|4 μL ‘E-pSB1A2 I13401-X
|25 μL
|25 μL
|-
|-
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|Ligation control
|Ligation control
|None
|None
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|4 μL ‘E-pSB1A2-I13401-X
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|4 μL ‘E-pSB1A2 I13401-X
|25 μL
|25 μL
|}
|}
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To all samples with an end volume of 25μL, 2.5 μL Ligase buffer was added.
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To all samples with an end volume of 25 μL, 2.5 μL Ligase buffer was added.
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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. The following concentrations of plasmid were obtained:
+
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"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
|'''BioBrick'''
|'''BioBrick'''

Revision as of 17:55, 1 August 2010

Lab work

Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing

The colony PCR of yesterday was put on1% agarose gel

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 [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 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)
[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
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