Team:NCTU Formosa/Notebook

About the notes

Our notes are the abbreviations of the experimental process, including titles, genes we transformed, plasmids we used, and resistances on the plasmids. We categorized these notes by date. Moreover, team members followed the iGEM protocol in each experimental step in order to get better experimental efficiency and the results we had expected.

Material and Methods

Minipreps of Plasmid DNA

We use Plasmid miniPREP kit (Genedirex Inc.) to obtain plasmid DNA for analysis, sequencing and cloning. Single colony is picked from LB agar plate and inoculated into LB medium with suitable antibiotics. Bacteria cells are harvest by centrifuge after incubation for 12 – 16 hours. Afterward, alkaline lysis is performed to release inside components, and DNA is purified by silicone resin-based column. The quality and amount of plasmid DNA is estimated by agarose gel electrophoresis stained by SyBr Safe (Invitrogen).

BioBrick assembly

To digest the desired DNA, appropriate restriction enzymes and reaction buffer (all perched from New England Biolabs) is used in 20 μL reaction volume. Upstream BioBrick is digested with EcoRI-HF and SpeI in CutSmart buffer. Downstream BioBrick and backbone part are digested with XbaI and PstI, EcoRI-HF and PstI respectively in NEBuffer 2. Reactions take place in 37 ℃ water bath for 2 hours or overnight.

Ligation is performed using BioBrick Assembly Kit Ligation Protocol. All the reagent and enzymes are perched from New England Biolabs). Reactions take place in 37 ℃ water bath for 2 hours or overnight in 16 ℃ incubator.

Transformation

DH5α E. coli competent cells (ECOS^TM 101, Yeastern Biotech Co., Ltd.) were used for the propagation of plasmid DNA. Standard transformation protocol is used: First place 2 μL plasmid or 10μL product into 1.5 mL mini centrifuge tube and chilled on ice. Competent cells are melt on ice before transformation. After adding 33 – 50 μL competent cells, heat shock at 42 ℃ for 1 minutes. Place cells into LB medium to recover then plating on LB agar plate and incubate for 16 – 18 hours.

PCR reaction

Taq DNA Polymerase 2x Master Mix Red (Ampliqon) and VF2 and VR primers (BBa_G00100 and BBa_G00101 respectively) are used for colony PCR. Single colony is picked and place into 10 μL PCR reagent, and followed by PCR with condition described below:

• Predenature: 95 ℃，10 min
• Denature, annealing and extension: [98 ℃, 10 s; 55 ℃, 30 s; 72 ℃, 1 m/kb + 30 s] × 25 cycles
• Hold: 4 ℃

Reagent and Medium

Lysogeny broth (LB): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl with with suitable antibiotics.

LB agar plate: LB medium with 1.5% (w/v) agar.

50X TAE buffer: tris base 242 g/L, glacial acetic acid 57.1 mL/L, 0.05 M EDTA, pH 8.0 Antibiotics working concentration:

• Kanamycin  : 25 μg/mL
• Ampicillin  : 100 μg/mL
• Chloramphenicol: 20 μg/mL

March 2013