Team:U of Guelph/Experiments
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
This year we prepared two different types of competent cells, DH5α and BL21. We used a chemical competency method involving calcium chloride. As the final step in preparation all cells were frozen in either 50 µl or 100 µl volumes by submersion in liquid nitrogen. Competent cells were then stored in a -80°C freezer for further use.
pET-28a isolation
pET-28a was purified from DH5α/pET-28a cells provided by Cezar Khursigara's Lab using a miniprep kit (Thermofisher Scientific). Purified DNA concentration was checked using a nanodrop and samples were frozen in a -20°C freezer for further use.
Site Directed Mutagenesis of pET-28a
PCR was used to conduct site directed mutagenesis to add a PstI cut site to pET-28a. Two basepairs were changed at the wild type XhoI cut site to change the sequence from ctcgag to ctgcag. Primers were ordered from IDT.
Confirmation of SDM Success
A double restriction enzyme digest was conducted to ensure that the PstI cut site had been added correctly. A single restriction enzyme digest and undigested plasmid were used as controls. All digestion products were run on an agarose gel and visualized using either Novel Juice (FroggaBio) or SyBr.
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
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
