Team:Illinois/MicA

From 2009.igem.org

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== '''MicA Target-GFP Fusion''' ==
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[https://2009.igem.org/Team:Illinois/sRNA_Library Back to sRNA Library Team page]
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'''Purpose:''' to test the efficiency of sRNA repression. This will be accomplished by forming two plasmids by ligating the target sequence of the sRNA onto the pXG-10 plasmid behind the reporter gene GFP and ligating the sRNA gene into the pJU-334 DNA strand. These plasmids will then be transformed into E. coli and the fluorescence measured with a plate reader.
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'''Protocol(s) Used:''' The protocol we used is that taken from Urban and Vogel, "A Green Fluorescent Protein (GFP)-Based Plasmid System to Study Post-Transcriptional Control of Gene Expression In Vivo"
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== '''MicA''' ==
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'''Recipe(s) Used:'''  
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'''Primers Used: for the sRNA gene we used
'''Primers Used: for the sRNA gene we used
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For the Target Sequence we used the primers from the Urban and Vogel paper: JV-0432 and JV-0433
For the Target Sequence we used the primers from the Urban and Vogel paper: JV-0432 and JV-0433
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== '''June 12''' ==
 
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Journal entry text goes here.
 
== '''June 16'''==
== '''June 16'''==

Latest revision as of 02:43, 22 October 2009

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MicA

Primers Used: for the sRNA gene we used Forward Primer: GAA AGA CGC GCA TTT GTT ATC 

Temp: (4*9) + (2*12) = 60 degrees

Second homology sequence: TTC CAG CCA CAC CGC AAA CGG ((TTCGGTATCA)) 

Reverse complement: CCG TTT GCG GTG TGG CTG G
Temp: (4*13) + (2*6) = 64 degrees
Cut site and overhang: GTTTTT TCTAGA

Reverse Primer: GTTTTT TCTAGA CCG TTT GCG GTG TGG CTG G

For the Target Sequence we used the primers from the Urban and Vogel paper: JV-0432 and JV-0433

June 16

We used PCR to extract the sRNA gene: MicA and target sequence : ompA. from the e.coli chromosome. We then ran a gel to make sure that we had the right DNA fragments. Our results corresponded to our predictions.

UI09Gel2.jpg

June 17

We are completing a digestion of MicA, OmpA, and OmpF. Following the digestion we will incubate with SAP and then run a gel to verify the digestion. We will then extract the DNA for the sRNA target sequence and sRNA gene.

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