Difference between revisions of "Team:Hamilton McMaster/Methodologies"

DNA

The majority of the DNA was provided by iGEM, either on the Plates 1-5 or by order. These included:

• 1C3-pbla
• T4 holin
• T4 endolysin
• cph8
• PompC-GFP
• pbad/araC
• ho1
• PcyA
• 1A3 backbone
• 1K3 backbone
• 1C3 backbone
• 1S3 backbone

The light sensing genes were isolated via PCR from a cassette purchased from Addgene. This cassette included the following genes:

• Ccas
• Ccar
• Pcpcg2

PCR

Because the DNA from the cassette, when isolated via PCR, would not have the usable cut sites (E X, and S P), the primers were designed with overhangs. Annealing temperatures were calculated in two stages, using the Warren A. Kibbe OligoCalc (Kibbe WA. 'OligoCalc: an online oligonucleotide properties calculator'. (2007) Nucleic Acids Res. 35(webserver issue): May 25.). The first stage only included the section of the forward and reverse primers that connected to the DNA in the cassette. The second stage included the entire length of the primers, as the overhangs had been formed.

Ccar PCR Thermocycle:

1. 95°C for 2min
2. 95°C for 30sec
3. 45°C for 30sec
4. 72°C for 1min
5. Repeat a-d for for 15 cycles
6. 95°C for 30sec
7. 56°C for 30sec
8. 72°C for 1min
9. Repeat f-i for 15 cycles
10. 4°C indefinitely

Ccas PCR Thermocycle:

1. 95°C for 2min
2. 95°C for 30sec
3. 45°C for 30sec
4. 72°C for 2:30min
5. Repeat a-d for for 15 cycles
6. 95°C for 30sec
7. 58°C for 30sec
8. 72°C for 2:30min
9. Repeat f-i for 15 cycles
10. 4°C indefinitely

Pcpcg2 PCR Thermocycle:

1. 95°C for 2min
2. 95°C for 30sec
3. 43°C for 30sec
4. 72°C for 45sec
5. Repeat a-d for for 15 cycles
6. 95°C for 30sec
7. 57°C for 30sec
8. 72°C for 45sec
9. Repeat f-i for 15 cycles
10. 4°C indefinitely

Cells

E. coli DH5α cells (Life Technologies / Invitrogen) were used for the entire transformation process.

Buffers and Enzymes for Digestion and Ligation

Buffers and enzymes (New England Biolabs):

• CutSmart
• NEBuffer 3.1
• EcoRI (E)
• Xbal (X)
• SpeI (S)
• PstI (P)
• T4 DNA ligase

Through parallel tests of all enzymes in all buffers, we found that CutSmart was the best buffer for all enzymatic digestions.

Mini-Prep

Mini-prep materials were bought from Life Technologies / Invitrogen. Mini-prep procedure followed standard protocol, involving centrifugation of cell samples into columns, and removal of supernatant. Mini-prep was followed by gel extraction.

Lab Equipment and Materials for Cell Cultures

 Materials including gel dyes, Red Safe, SOC and LB media, and antibiotics (Ampicillin, Chloramphenicol, Kanamycin, and Streptomycin) were bought from Life Technologies / Invitrogen, and other materials and lab equipment were graciously provided by the allure. and McMaster undergraduate Cell Biology lab.

Gel Visualization and Gel Extraction

Gels were made using 1% agarose and Red Safe for visualization. Gel visualization was performed using the BioRad Gel Doc EZ Reader. Gel extraction was completed using standard razor blades on a UV light box.

Digestion and Ligation

The 3A assembly method was used to assemble the plasmids, as recommended by iGEM HQ, and is represented in the figure below:

Diagram representing the 3A Assembly. Source: iGEM.

Growth and Transformation

Bacterial growth was accomplished in LB either on agar plates or tubes, with their respective antibiotics. Transformations were accomplished by the standard heat-shock method at 85°C.

Sequencing of gel-verified and transformed plasmids