Reporter: Week 10 July 17-23

From 2011.igem.org

(Difference between revisions)
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==Monday, July 18==
==Monday, July 18==
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The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.
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Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.
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{|border="1"
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!Protocol
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|colspan="2" align="center"|'''Part Involved with Protocol'''
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|-
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|Insert PCR (extension time=1:15)
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|colspan="2"|GFP
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|-
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|rowspan="2"|Restriction Digest
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|Insert using NgoMIV and PstI (buffer 1)
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|GFP
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|-
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|Vector using AgeI and PstI (buffer 1)
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|-
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|Ligation
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|colspan="2"|tev cleavage site+GFP
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|-
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|Transformation/Plating
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|colspan="2"|The above ligation was transformed into<br />Escherichia coli cells and plated on<br />kanamycin resistant plates.
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|}
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The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.
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The GusA and LacZ&alpha; genes were cloned into K3 using the 10AA linker+K3 as the vector.
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==
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[[Team:Penn_State/Notebook| Back to Notebook]]
[[Team:Penn_State/Notebook| Back to Notebook]]

Revision as of 17:17, 2 August 2011

Sunday, July 17

No colonies grew for tev cleavage site+GFP, and only one colony grew for cI cleavage site+GFP. This colony was amplified through colony PCR (extension time=1:20). The PCR products were run on an agarose gel, which yielded a band that corresponded to around 1000 bp, which was expected. The colony was grown up in culture overnight.

Monday, July 18

The tev mutagenesis and cI cleavage site+GFP plasmids were sent to the sequencing center. The sequencing results showed that the cI cleavage site+GFP worked, but the tev mutagenesis did not.

Since we have had trouble inserting the cleavage sites in front of the GFP gene, the GFP was inserted behind the cleavage sites.

Protocol Part Involved with Protocol
Insert PCR (extension time=1:15) GFP
Restriction Digest Insert using NgoMIV and PstI (buffer 1) GFP
Vector using AgeI and PstI (buffer 1)
Ligation tev cleavage site+GFP
Transformation/Plating The above ligation was transformed into
Escherichia coli cells and plated on
kanamycin resistant plates.

The promoter and RBS construct (J23100+B0034) was placed in front of GFP+tev cleavage site, XylE+small linker and XylE+Imp linker following the procedure performed on Thursday, July 7.

The GusA and LacZα genes were cloned into K3 using the 10AA linker+K3 as the vector.

==


Back to Notebook

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