Team:Illinois/MicF

From 2009.igem.org

(Difference between revisions)
(MicF Target-GFP Fusion)
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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

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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.

UI09Gel2.jpg

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.

MicF OmpF digests.jpg