Haynes:TypeIIS Assembly: Difference between revisions
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| [[Image:Haynes_TIIS_fig2.png|250px|Figure 2]] || Design a pair of primers to add ''BsmBI'' sites to the ends of a vector backbone. The "cacacca" before ''BsmBI'' is used to help restriction enzyme positioning. The "a" after ''BsmBI'' is a spacer that is required to generate a correct 4-base sticky end. <br> | | [[Image:Haynes_TIIS_fig2.png|250px|Figure 2]] || Design a pair of primers to add ''BsmBI'' sites to the ends of a vector backbone. The "cacacca" before ''BsmBI'' is used to help restriction enzyme positioning. The "a" after ''BsmBI'' is a spacer that is required to generate a correct 4-base sticky end. <br> | ||
'''Vector Primers''' | '''Vector Primers''' | ||
* Forward Primer: 5'-<font color=#666666>cacacca</font>'''CGTCTCa''' + <font color=#660099>15 bp of " | * Forward Primer: 5'-<font color=#666666>cacacca</font>'''CGTCTCa''' + <font color=#660099>15 bp of "Vector right" top strand</font> | ||
* Reverse Primer: 5'-<font color=#666666>cacacca</font>'''CGTCTCa''' + <font color=#660099>15 bp of " | * Reverse Primer: 5'-<font color=#666666>cacacca</font>'''CGTCTCa''' + <font color=#660099>15 bp of "Vector left", bottom strand</font><br> | ||
'''pSB1A3 Vector Primers''' - already available in the Haynes lab freezer | '''pSB1A3 Vector Primers''' - already available in the Haynes lab freezer | ||
* Forward Primer gg0001: 5'-<font color=#666666>cacacca</font>'''CGTCTCa'''<font color=#660099>actagtagcggccgct</font> | * Forward Primer gg0001: 5'-<font color=#666666>cacacca</font>'''CGTCTCa'''<font color=#660099>actagtagcggccgct</font> | ||
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| [[Image:Haynes_TIIS_fig3.png|250px|Figure 3]] || '''Part A Primers''' | | [[Image:Haynes_TIIS_fig3.png|250px|Figure 3]] || '''Part A Primers''' | ||
* Forward Primer: 5'-<font color=#666666>cacacca</font> | * Forward Primer: 5'-'''<font color=#666666>cacacca</font>CGTCTCa + <font color=#660099>4 bp of "Vector left" top strand</font> + <font color=#ff6600>15 bp of "Part A" top strand</font>'''<br> | ||
* Reverse Primer: 5'- | Note: For insertion into pSB1A3, "last 4 bp of vector left" = <font color=#660099>'''TAGA'''</font> | ||
* Reverse Primer: 5'-'''<font color=#666666>cacacca</font>CGTCTCa + <font color=#660099>15 bp of "Part A" bottom strand<br> | |||
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| [[Image:Haynes_TIIS_fig4.png|250px|Figure 5]] || ''' Part B Primers''' | | [[Image:Haynes_TIIS_fig4.png|250px|Figure 5]] || ''' Part B Primers''' |
Revision as of 16:37, 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.
- Use the following formula to calculate the volume of purified DNA (x) you will need to dilute in a final volume of 20 μL
- Formula: x = 20 μL final volume * 20 fmols/μL * length in bp * 650 fg/fmol ÷ 1,000,000 fg/ng ÷ measured ng/μL
2. Golden Gate Reaction
Reagent | Vol. |
20 fmol of each DNA part | up to 8.0 |
10x T4 ligase buffer (Promega) | 1.0 |
T4 ligase (NEB) | 0.25 |
BsmBI | 0.5 |
dH2O | 0.25 |
10.0 μL |
Thermal cycling
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
3. Transformation
- 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