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.<br
   

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; 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 15 minutes

1. Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
   X ng insert = (bp insert / bp vector) x 2 x 50 ng vector
2. Calculate how many μL of insert and vector you will need for each ligation:
   X μL insert = desired ng insert ÷ insert concentration ng/μL (do the same for vector)
3. Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown below:

	Ligation	Negative Control
Insert DNA (X ng)	___ μL	none
Vector DNA (50 ng)	___ μL	same
2x Roche Rapid Ligation buffer	5.0 μl	same
NEB T4 ligase	1.0 μl	same
dH2O	___ μL	___ μL + insert volume
	10.0 μL total	same
Mix the reaction(s) thoroughly by flicking the tube. Incubate at room temperature for 10 minutes.

Transform bacteria with the ligated plasmids 30 minutes

1. Warm selection agar plates at 37°C.
2. Incubate DH5α Turbo competent cells on ice just until thawed. Use 30 μL per ligation.
3. Add 30 μL thawed cells to the ligation reaction. Immediately place on ice and incubate for 10 min. (Do not heat shock; No 30 min. recovery is required for Amp resistance)
4. Label the pre-warmed plates with the antibiotic name, strain name, ligation (e.g., "BB part A insert + BB part B vector"), your initials, and the date.
5. Pipette the total volume of cells + ligation onto the agar; spread using sterile glass beads.
6. Incubate overnight at 37°C to get colonies

Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.

Day 3: Confirm the assembly

Check the plates, grow cultures, and do minipreps 6 hours

1. Compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies.
2. If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures (see Day 1, Grow liquid cultures). Grow for 5 - 6 hours.
   If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation.
3. Miniprep the DNA from the liquid cultures (see Day 2, Extract the plasmid DNA: Qiagen Miniprep Kit)
4. Digest 2 uL of each DNA sample with EcoRI/ PstI and check via gel electrophoresis (1% agarose) to confirm the assembled construct size. You should see one fragment that is the backbone, and another fragment that equals the total size of the two BioBrick parts you assembled.

Timeline

• Level 1, Newbie: Undergraduates and unseasoned scientists can expect to spend a week to two weeks on one assembly step. You will inevitably spill something, forget a step, plan an assembly incorrectly, or mess up some other inventive way. Or you have classes and can't spend every day in the lab.
• Level 2, Graduate Student: Typically have experience pipetting and handling samples well and can expect to spend 3 days per assembly. If you have no life and are super-ambitious, you can crank out an assembly cycle in two days (when Day 2 procedures are started immediately after the Day 3 procedures in a single day), and complete three assemblies in one week.
• Level 3, Postdoc "Cloning Ninja": If you have no life, are super-impatient, and are trying to publish papers, you can crank out an assembly cycle in two days, and complete three assemblies in one week.