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.

Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.

Make streaks from glycerol stocks 6 hours

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 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 1.5 hours
To extract the plasmid DNA from the bacteria, perform a mini prep (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 with restriction enzymes 30 minutes

1. 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
2. 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 buffer + green loading dye	3.0 μl
dH2O	10.0 μl
	30.0 μl total
*For low yield DNA, use up to 25 μL and decrease dH2O accordingly. Mix the reaction(s) thoroughly by flicking the tube. Incubate at 37°C for 10 minutes.

Separate the fragments via gel electrophoresis and purify the fragments 2 hours

1. Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask.
2. Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
3. Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
4. Add 5 μl of 10 mg/ml ethidium bromide (etBr) to the agarose for a final concentration of ~0.8 μg/mL etBr. Mix by swirling (avoid making bubbles).
5. Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
6. Fill a gel electrophoresis chamber with 1x TAE.
7. Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
8. Carefully pipette 15 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
9. Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
10. Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
11. Remove the gel from the chamber and photograph under UV light.
12. Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA (refer to the Qiagen gel purification protocol; elute with 30 μL EB buffer).
13. Measure the concentration of the purified fragment samples with a Nanodrop Spectrophotometer.
14. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.

Ligate (paste) the DNA fragments together