Haynes:TypeIIS Assembly

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'''pSB1A3 Primers''' - already available in the Haynes lab freezer
'''pSB1A3 Primers''' - already available in the Haynes lab freezer
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* Forward Primer: 5'-
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* Forward Primer: 5'-cacaccaCGTCTCAactagtagcggccgct
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* Reverse Primer: 5'-
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* Reverse Primer: 5'-cacaccaCGTCTCAtctagatgcggccgcg
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|-
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Part A Primers
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| image || Part A Primers
* Forward Primer: 5'-cacaccaCGTCTCa + last 4 bp of "vector left" (top strand) + first 15 bp of Part A (top strand)
* Forward Primer: 5'-cacaccaCGTCTCa + last 4 bp of "vector left" (top strand) + first 15 bp of Part A (top strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part B, reverse complement (bottom strand) + last 15 bp of Part A, reverse complement (bottom strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part B, reverse complement (bottom strand) + last 15 bp of Part A, reverse complement (bottom strand)
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+
|-
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Part B Primers
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| image || Part B Primers
* Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part B (top strand)
* Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part B (top strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part C, reverse complement (bottom strand) + last 15 bp of Part B, reverse complement (bottom strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part C, reverse complement (bottom strand) + last 15 bp of Part B, reverse complement (bottom strand)
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+
|-
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Part C Primers
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| image || Part C Primers
* Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part C (top strand)
* Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part C (top strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of "vector right", reverse complement (bottom strand) + last 15 bp of Part C, reverse complement (bottom strand)
* Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of "vector right", reverse complement (bottom strand) + last 15 bp of Part C, reverse complement (bottom strand)
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| Purify the PCR products using a Zymo clean and Concentrator kit.
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| image || Purify the PCR products using a Zymo clean and Concentrator kit.
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Revision as of 20:38, 18 January 2013

<- Back to Protocols

Type IIS Assembly

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 (CGTCTCnnnnn/).


Prepare the parts with PCR

  • 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!
image First, map out the assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.
image Design a pair of primers to add BsmBI sites and (in some cases) complementary overlaps to each end...

Vector Primers

  • Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of "vector right" (top strand)
  • Reverse Primer: 5'-cacaccaCGTCTCa + last 15 bp of "vector left", reverse complement (bottom strand)

pSB1A3 Primers - already available in the Haynes lab freezer

  • Forward Primer: 5'-cacaccaCGTCTCAactagtagcggccgct
  • Reverse Primer: 5'-cacaccaCGTCTCAtctagatgcggccgcg
image Part A Primers
  • Forward Primer: 5'-cacaccaCGTCTCa + last 4 bp of "vector left" (top strand) + first 15 bp of Part A (top strand)
  • Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part B, reverse complement (bottom strand) + last 15 bp of Part A, reverse complement (bottom strand)
image Part B Primers
  • Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part B (top strand)
  • Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of Part C, reverse complement (bottom strand) + last 15 bp of Part B, reverse complement (bottom strand)
image Part C Primers
  • Forward Primer: 5'-cacaccaCGTCTCa + first 15 bp of Part C (top strand)
  • Reverse Primer: 5'-cacaccaCGTCTCa + first 4 bp of "vector right", reverse complement (bottom strand) + last 15 bp of Part C, reverse complement (bottom strand)
image Purify the PCR products using a Zymo clean and Concentrator kit.

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.


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
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