Model Procedure for Assembling Parts: Classic Ligation for Beginners
or, Cloning Sensei's Guide For the Aspiring Cloning Ninja
Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.
Make streaks from glycerol stocks 6 hours
- 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 or plastic micropipette tip 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 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|
|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
- 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 to the right:
Separate the fragments via gel electrophoresis and purify the fragments 2 hours File:KAH 011711 gel2.tif
- Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask.
- 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.
- Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
- Add 6 μl of SYBR safe stain. Mix by swirling (avoid making bubbles).
- Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
- Fill a gel electrophoresis chamber with 1x TAE.
- Gently remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
- 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.
- 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.
- Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
- Remove the gel from the chamber and photograph under UV light.
- 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).
- 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.
|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
- 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
- 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
- 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.
- Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
- Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
- Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
- 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.
- Incubate on ice for 10 min.
- 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.
- Add 750 μL sterile SOC medium to each sample.
- 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.
- Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
- Pellet the cells by centrifugation at top speed for 3 minutes.
- Discard the SOC supernatant.
- Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
- Pipette the total volume of cells onto the agar; spread using sterile glass beads.
- 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
- Compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies.
- 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.
- Miniprep the DNA from the liquid cultures (see Day 2, Extract the plasmid DNA: Qiagen Miniprep Kit)
- 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.