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:
New Construct Name: BioBrick Insert Name, size (bp), cut sites + BioBrick Vector Name, size+backbone (bp), cut sites
- Set up your digest reaction(s) as shown below:
|Plasmid DNA||15.0 μl*|
|Fermentas FastDigest enzyme 1||1.0 μl|
|Fermentas FastDigest enzyme 2||1.0 μl|
|10x FastDigest green buffer||3.0 μl|
|30.0 μl total|