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| || Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final transformation step. Check 10 μL of the reaction on and agarose gel. Purify the remaining 40 μL of PCR products using a Zymo clean and Concentrator kit (or similar PCR clean up kit).
Revision as of 20:18, 19 January 2013
by Karmella Haynes, 2013
Principle: The familiar "BioBrick cloning" enzymes (i.e., EcoRI, NotI, XbaI, SpeI, PstI) are Type II restriction enzymes, which cut the sequences that they specifically bind to. The Type IIS Assembly method uses a Type IIS restriction enzyme, which binds at a specific sequence and cuts at a non-specific location exactly five base pairs away. As a result, the enzyme cleaves away its own binding site and leaves behind the most useful feature of assembly, sticky overhangs. When designed properly, Type IIS sites can be used to perform seamless assembly of parts. As an added convenience, this protocol allows cutting and ligation to occur in a single tube, as a single reaction. Thus, gel purification steps can be eliminated.
This protocol uses the Type IIS restriction enzyme BsmBI (CGTCTCn/nnnn).
Use PCR to prepare the parts
- Multiple parts can be assembled in one step.
- Parts and the destination vector should be amplified by PCR.
- Make sure that none of the parts/ vector have any BsmBI sites!
Digestion/ Ligation Reaction
1. Dilute the purified PCR product to 20 fmol/μL
- Measure ng/μL of the purified sample.
- The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
- Formula: x = length in bp ÷ measured ng/μL * 0.26
2. Golden Gate Reaction
|20 fmol of each DNA part||up to 8.0|
|10x T4 ligase buffer (Promega)||1.0|
|T4 ligase (NEB)||0.25|
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
- Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
- Incubate on ice for 2 min., heat shock at 42°C for exactly 90 sec., immediately place on ice.
- Add 800 μL sterile SOC medium.
- Grow with shaking at 37°C for 30 min.
- Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.
- Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.
- Plate cells on pre-warmed LB agar + antibiotic. Grow overnight at 37°C.
- Quick-transormation (e.g., DH5α-Turbo) is not recommended