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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 or plastic micropipette tip 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).

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.

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 to the right:

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

Blue dashed lines border where the gel was cut to excise vector fragments (lanes 1 and 2) and insert fragments (lanes 3 and 4).
  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 6 μl of SYBR safe stain. 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. Gently remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
  8. Carefully pipette 3-6 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. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.

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

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
    X μL vector = 50 ng vector ÷ vector concentration ng/μL
  3. Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown here:

Transform bacteria with the ligated plasmids 60 minutes

We'll be following the traditional transformation protocol described here.

  1. Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
  2. Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
  3. Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
  4. Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down gently to mix the cells and DNA.
  5. Incubate on ice for 10 min.
  6. Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
  7. Add 750 μL sterile SOC medium to each sample.
  8. Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
  9. Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
  10. Pellet the cells by centrifugation at top speed for 3 minutes.
  11. Discard the SOC supernatant.
  12. Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
  13. Pipette the total volume of cells onto the agar; spread using sterile glass beads.
  14. Incubate the inverted plate(s) overnight at 37°C to get colonies.

Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted). 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.


  • 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.