Difference between revisions of "Haynes:Assembly101"
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Revision as of 17:37, 20 January 2012
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
- 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 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).
Digest (cut) the DNA 30 minutes Use the appropriate restriction enzymes to cut each BioBrick plasmid
- 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 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|
|30.0 μl total|
|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
- 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 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).
- Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
- Fill a gel electrophoresis chamber with 1x TAE.
- Remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
- Carefully pipette 15 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 feel at 100 - 110 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).