Model Procedure for Assembling Parts: Classic Ligation for Beginners
or, Cloning Sensei's Guide For the Aspiring Cloning Ninja
A. Growing a Bacterial (E. coli) Culture
Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.
Make streaks from glycerol stocks
- Warm an agar plate at 37°C for at least 20 min.
- Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date.
- Locate the desired -80°C glycerol stock. Use a sterile wooden toothpick to scrape up a tiny bit of the frozen bacteria and streak the plate.
- Incubate the plate at 37°C for 6 hours to grow the bacteria.
Grow liquid cultures
- Label 15 ml sterile culture tube(s) appropriately. Fill each tube with 2 ml of LB growth medium + appropriate antibiotic (e.g., 100 μg/ml ampicillin).
- Using a sterile pipette tip, touch the bacterial streak (or pick up a single colony) and put the tip into the LB medium (bacterial end down).
- Grow the cultures overnight in a shaking 37°C incubator.
Day 2: Extract the plasmids. Digest (cut), purify, and ligate (paste) the BioBricks. Put the assembled plasmid into bacteria
Extract the plasmid DNA: Qiagen Miniprep Kit
To extract the plasmid DNA from the bacteria, perform a mini prep (~1.5 hours; refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl).
Digest (cut) the DNA Use the appropriate restriction enzymes to cut each BioBrick plasmid
- First, write out a brief assembly strategy: What are you building? What is the name and size of the BioBrick insert? What is the name and size of the BioBrick vector (plus backbone)? What will the insert and vector be cut with?
- Set up your digest reaction(s) as shown below:
|Plasmid DNA||___ μl|
|Fermentas FastDigest enzyme 1|| ___ μl
Enzyme 2 __ μl buffer 1.0 μl dH2O __ μl 50 μl