Experiments

Experiment Overview

Our experimental goal this year was to clone frc and oxc into E. coli DH5α using pET-28a. To accomplish this goal we had frc and oxc synthesized by IDT and used PCR to add the correct restriction enzyme sites. Site directed mutagenesis was used to add the PstI cut site to pET-28a, then frc and oxc were inserted using standard restriction enzyme digests and ligation. pET-28afrc and pET-28aoxc were then transformed into DH5α.

Preparation of Competent Cells

Here include information on both the DH5a and BL21 competent cell prep.

pET-28a isolation

Here include information how we isolated and purrified pET-28a from DH5a/pET-28a

Site Directed Mutagenesis of pET-28a

Here include information on how we mutated pET-28a to insert PstI RE site.

Confirmation of SDM Success

Here include information on the steps we took to confirm that our SDM occurred correctly.

Isolation of pET-28a(PstI) from DH5a

Here include information on how we isolated and purrified pET-28a(PstI) from DH5a/pET-28a

Addition of EcoRI and PstI to FRC and OXC

Here include information on how we designed FRC and OXC, ordered them, designed primers and added the RE sites and how we checked that it had worked

Adding FRC and Oxc to pET-28a(PstI)

Here include information on how we RE digested FRC, OXC and pET-28a and preformed two separate ligations.

Transformation of pET-28a(PstI)frc and pET-28a(PstI)oxc into DH5a

Here include information on how we transformed FRC and OXC into DH5a

Isolation of pET-28a(PstI)frc and pET-28a(PstI)oxc from DH5a

Here include information on how we isolated and purrified pET-28a(PstI)frc and pET-28a(PstI)oxc from DH5a

Confermation of the Presence of frc and oxc

Here include information on how we confirmed the presence of frc and oxc

Transformation of pET-28a(PstI)frc and pET-28a(PstI)oxc into BL21

Here include information on how we transformed pET-28a(PstI)frc and pET-28a(PstI)oxc into BL21

Confermation of the Presence of frc and oxc in BL21

Here include information on how we confirmed the presence of frc and oxc in BL21

Expression of FRC and OXC

Here include information on how we expressed FRC and OXC

Isolation of FRC and OXC

Here include information on how we isolated FRC and OXC

Confirmation of the Presence of FRC and OXC

Here include information on how we confirmed the presence of FRC and OXC

Protocols

Here we will include a detailed list of all the basic protocols used in our experiments. Specific parameters for things such as PCRs will be noted above

Competent Cells

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Plasmid Isolation

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Agarose Gel

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PCR

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PCR Purification

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Restriction Enzyme Digest

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Spread Plating

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Streak Plating

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Liquid Culture Inoculation

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Transformation

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Refrences

Note here we have to include references to places where we pulled our protocols including lab methods.

Rose, D. This is a test (2017). Sci. Awesome. 28-29