Haynes:Assembly101

Model Procedure for Assembling Parts: Classic Ligation for Beginners
or, Cloning Sensei's Guide For the Aspiring Cloning Ninja

by Karmella Haynes, 2012

When I was a postdoc in Pam Silver's lab at Harvard (2008 - 2011), my lab mates and I generated large numbers of BioBrick assemblies so rapidly, and perhaps stealthily, that one of our colleagues in the department referred to us as "cloning ninjas." This guide is based on the MIT Registry of Standard Biological Parts suggested approach, which I've modified to make ligation-based assembly as quick and painless as possible. Let's begin.

A. Growing a Bacterial (E. coli) Culture

Escherichia coli (K-12 and related strains) is a common lab strain that is used for molecular cloning. BioBrick plasmids are usually stored in E. coli that are suspended in a glycerol solution and frozen at -80°C. When E. coli are grown at 37°C, they divide once about every 20 minutes. As the bacteria multiply via mitosis, they are making more copies of the plasmid you want. I use fast-growing strain DH5α Turbo to speed up the process.

Day 1*:
Make streaks from glycerol stocks

1. Warm an agar plate at 37°C for at least 20 min.
2. Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date.
3. 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.
4. Incubate the plate at 37°C for 6 hours to grow the bacteria.
   

Grow liquid cultures

1. 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).
2. 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).
3. Grow the cultures overnight in a shaking 37°C incubator.
   
   

*This may take two days instead of one if you're starting with a slow-growing strain.

Day 3:
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

1. 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?
2. 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