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Team:Illinois/MicF
From 2009.igem.org
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*Forward OmpF (sRNA gene) primer: GTTTTTATGCATAGACACATAAAGACACCAAACTC | *Forward OmpF (sRNA gene) primer: GTTTTTATGCATAGACACATAAAGACACCAAACTC | ||
*Reverse OmpF (sRNA gene) primer: GTTTTTGCTAGCAGGGACGATCACTG | *Reverse OmpF (sRNA gene) primer: GTTTTTGCTAGCAGGGACGATCACTG | ||
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| + | MicF gene sequence: *GCTATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTACCCCTATTTCAACCGGATGCCTCGCATTCGGTTTTTTTTACCCTTCTTTACACACTTTTCATTATTCTG | ||
== '''June 16'''== | == '''June 16'''== | ||
Revision as of 23:15, 17 June 2009
MicF Target-GFP Fusion
Purpose: MicF will be fused to the target sequence from the OmpF gene to the GFP gene on a low-copy plasmid and transform this into E. coli cells along with a high-copy plasmid carrying the MicF gene. We expect to see translational repression of GFP by the small RNA MicF.
Protocol(s) Used: (links to protocols page)
Recipe(s) Used:
Primers Used: (5' -> 3')
- Forward MicF (sRNA gene) primer: 5'p+GCTATCATCATTAACTTTATTTATTACCG
- Reverse MicF (sRNA gene) primer: GTTTTTTCTAGAGGTAGCACAGAATAATGAAA
- Forward OmpF (sRNA gene) primer: GTTTTTATGCATAGACACATAAAGACACCAAACTC
- Reverse OmpF (sRNA gene) primer: GTTTTTGCTAGCAGGGACGATCACTG
MicF gene sequence: *GCTATCATCATTAACTTTATTTATTACCGTCATTCATTTCTGAATGTCTGTTTACCCCTATTTCAACCGGATGCCTCGCATTCGGTTTTTTTTACCCTTCTTTACACACTTTTCATTATTCTG
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.
June 17
We are completing a digestion of MicF and OmpF. Following the digestion we will incubate with Shrimp Alkanline Phosphatase and then run a gel to verify the digestion. We will then extract the DNA for the sRNA target sequence and sRNA gene.
