Haynes:TypeIIS Assembly: Difference between revisions
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* Reverse Primer: 5'-'''<font color=#666666>cacacca</font>CGTCTCa + <font color=#660099>15 bp of "Vector left" bottom strand</font>'''<br> | * 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> | * Forward Primer gg0001: 5'-'''<font color=#666666>cacacca</font>CGTCTCa<font color=#660099>actagtagcggccgctgcag</font>''' | ||
* Reverse Primer gg0002: 5'-'''<font color=#666666>cacacca</font>CGTCTCa<font color=#660099> | * Reverse Primer gg0002: 5'-'''<font color=#666666>cacacca</font>CGTCTCa<font color=#660099>tctagaagcggccgcgaattcc</font>''' | ||
<br> | <br> | ||
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| <br>'''Dilute the purified PCR product to 20 fmol/μL''' | | <br>'''Dilute the purified PCR product to 20 fmol/μL''' | ||
* Measure ng/μL of the purified sample. | * 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 = | * The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = '''length in bp''' ÷ '''measured ng/μL''' * ''20 fmols/μL * 650 fg/fmol ÷ 1,000,000 fg/ng'' * 20 μL final volume | ||
** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * 0. | ** Formula: x = '''length in bp''' ÷ '''measured ng/μL''' * ''0.013'' * 20 | ||
** Note: final volume can be changed by changing the last number from 20 to something else. | |||
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| [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]] | | [[Image:Haynes_TIIS_fig7.png|250px|Figure 7]] |
Revision as of 18:13, 12 September 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).
Bacterial 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 45 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