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 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. Samples were frozen in a -20°C freezer for further use.
Isolation of pET-28a(PstI) from DH5a
pET-28a was purified from DH5α/pET-28a cells 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.
Addition of EcoRI and PstI to frc and oxc
PCR was used to add EcoRI and PstI cut sites to the ends of frc and oxc. PCR primers and synthesized frc and oxc were all obtained from IDT.
Adding frc and oxc to pET-28a
The genes frc and oxc were added individually to pET-28a using standard ligation techniques.
Transformation of pET-28afrc and pET-28aoxc into DH5α
Using standard heat shock techniques pET-28afrc and pET-28aoxc were transformed into DH5α. Transformed cells were incubated for 1 hour at 37°C with rotation (using our homemade rotator) then plated on Kanamycin plates.
Confirmation of the Presence of frc and oxc
Colony PCR was used to confirm the presence of frc and oxc from the colonies grown from the transformation. One of each type of colony was selected to make liquid culture, and then glycerol stocks from for long term storage of the transformed bacteria.