# Haynes:TypeIIS Assembly

(Difference between revisions)
 Revision as of 19:57, 9 January 2013 (view source)← Previous diff Revision as of 20:08, 9 January 2013 (view source)Next diff → Line 47: Line 47: * Use the following formula to calculate the volume of purified DNA you will need to dilute in a final volume of 20 μL * Use the following formula to calculate the volume of purified DNA 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''' ** Formula: x =  ('''20''' μL final volume * '''20''' fmols/μL * '''length in bp''' * '''650''' fg/fmol ÷ '''1,000,000''' fg/ng  ÷ '''measured ng/μL''' - * Make 20 μL of dilution - # Dilute each product to ~20 fmol/μL + - pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 + 2. Golden Gate Reaction + + {| {{table}} + |- + | 20 fmol of each DNA part || up to + |- + | 10x T4 ligase buffer (Promega) || 1.0 + |- + | T4 ligase (NEB) || 0.25 + |- + | BsmBI || 0.5 + |- + | dH2O || --- + |- + |   || 10.0 μL + |} + + Thermal cycling + * [45°C, 2min; 16°C 5min] x25 + * 60°C 10min + * 80°C 20min

## Revision as of 20:08, 9 January 2013

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 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/).

Part Preparation - 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!

1. First, map out the assembly. For instance: vector left - Part A - Part B - Part C - vector right.
In this example, the vector, Part A, Part B, and Part C will be PCR amplified.
2. Design a pair of primers for each part and the vector...

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)

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)

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)

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)

3. 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 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

 20 fmol of each DNA part up to 10x T4 ligase buffer (Promega) 1.0 T4 ligase (NEB) 0.25 BsmBI 0.5 dH2O --- 10.0 μL

Thermal cycling

• [45°C, 2min; 16°C 5min] x25
• 60°C 10min
• 80°C 20min