Gibson Assembly® – Construct DesignGuidance for Cloning one Insert into a Vector

Complete product information and additional resources are available at sgidna.com.

To assemble an insert with a vector, a 20–40 bp homologous region must be added to the ends of the fragment(s). The homologous region sequence may be derived from the vector (Scenario 1), from the insert (Scenario 2), or from both the vector and insert ends (not shown here). For guidance on multiple fragment assembly, refer to the Gibson Assembly® HiFi 1 Step and Ultra product manuals at sgidna.com/product_documentation.

Design Strategies for Gibson Assembly® Seamless Cloning

+Insert

Vector

Scenario 1− Scenario 2−

If PCR amplifying the insert is preferred If PCR amplifying the vector is preferredAdd sequence homologous to the vector to the

ends of the insertAdd sequence homologous to the insert to the

ends of the linearized vector

+ +

Considerations Considerations• For small inserts (< 2 kb)• Using one vector for cloning different inserts

(such as shotgun cloning or preparing combinatorial libraries)

• For small vectors (< 8 kb)• Wheninsertsarelargeordifficulttoamplify• Using one insert to shuttle between different

vectors

Technical Tips•Gel purify DNA fragments prior to performing the assembly reaction.

• The cloning vector can be linearized by restriction enzyme digest. Blunt ends, 5’ overhangs, and 3’ overhangs are all compatible with Gibson Assembly®cloningwithoutfurthermodification.

• The length of the homologous ends depends on the complexity of the assembly reaction. Typical factors affecting complexity are the length and number of DNA fragments. As complexity increases, increase the length of the homologous ends. Long homologous ends increase the success rate of assembly.

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2

Scenario 1: Adding vector sequence to the ends of the insert

Vector Insert

Prepare by Restriction Enzyme Digest or PCR Amplification Prepare using PCR Amplification

Restriction Enzyme Digestion

1. Digest vector with restriction enzyme(s).

2. Purifythevectorusinggelpurification(preferred)orcolumnpurification.

or

PCR Amplification

1. Identify cloning junction.

Insertion site

2. Select primers for PCR.

• 20–30 nucleotides

• Tm optimized, preferred5’ 3’

Primer-Fwd

5’3’

Primer-Rev

3. Amplify the vector.

4. (Optional) Treat the vector reaction with DpnIfollowingamplificationtominimizebackground.

1. Identify the insert sequence intended for cloning.

Desired Sequenceextraneous extraneous

2. Select 20 nt of sequence at 5’ and 3’ insert ends.

20 nt5’ 3’

20 nt3’ 5’

3. Select the vector sequence you will add to insert.

5’ 3’

5’3’

40 bp vector homologous sequence to add to the insert

40 bp vector homologous sequence to add to the insert

4. Create primers to be used for adding vector sequence to the ends of the insert by cutting and pasting the selected sequences together.

5’ 3’40 nt vector 20 nt

insert3’ 5’

20 nt insert 40 nt vector

5. (Optional) Add a restriction enzyme recognition site at the vector/insert junction to facilitate downstream screening.

5’ 3’40 nt vector

20 nt insert

3’ 5’

RE site 40 nt vector

20 nt insert

RE site

6. Amplify the insert.

Result

Linearized Vector +

Insert Homologous endHomologous end

RE site RE site

3

Scenario 2: Adding insert sequence to the ends of the vector

Prepare Vector by PCR AmplificationPrepare the Insert by Restriction Enzyme Digest,

PCR Amplification, or by Synthesis

Restriction Enzyme Digestion

1. Digest the insert with restriction enzyme(s).

2. Purifytheinsertusinggelpurification(preferred)orcolumnpurification.

or

PCR Amplification

1. Select primers for PCR.

• 20–30 nucleotides

• Tm optimized, preferred

5’

3’

20−30 nt

20−30 nt

2. Amplify the insert.

or

DNA Synthesis

Synthetic DNA, such as SGI-DNA Tiles™, can be incorporated into a vector.

1. Identify the vector cloning junction.

Insertion site

2. Selectamplificationsequencesatthejunction.

5’ 3’

20 nt vector sequence

5’3’20 nt vector sequence

3. Select the insert sequence you will add to the vector.

5’3’40 nt

40 nt3’5’

4. Create primers to be used for adding insert sequence to the ends of the vector by cutting and pasting the selected sequences together.

5’ 3’40 nt insert

20 nt vector

5’3’

20 nt vector

40 nt insert

5. (Optional) Add a restriction enzyme recognition site at the vector/insert junction to facilitate downstream screening.

5’5’

3’3’

RE recognition site

RE recognition site40 nt insert

40 nt insert

20 nt vector

20 nt vector

6. Amplify the vector.

Result

Linearized Vector

Homologous ends with optional RE recognition sites shown

+

Insert

Technical Services For technical assistance, please contact technical services at techservices@sgidna.com.

Trademark Information Synthetic Genomics® and Gibson Assembly® are registered trademarks and DNA Tiles™ is a trademark of Synthetic Genomics Inc.

Phusion®isaregisteredtrademarkofThermoFisherScientificInc.

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ProceduresLinearize the Vector by Restriction Enzyme Digestion (Scenario 1 Only)

Employ a restriction enzyme digest method that will allow for complete digestion of the vector (for example, use a double-digest). Gel extract the linearized vector to minimize vector background and reduce the number of observed background colonies before proceeding to the assembly reaction.

PCR Procedure for amplifying DNA fragments (Scenario 1 and 2)

After selecting your Design Strategy and designing your primers, amplify your fragments with PCR.

Scenario 1 Scenario 2

Insert Optional (may use restriction enzyme digestion)

Vector Optional (use if not linearizing by restriction enzyme

digestion)

Note:FollowingPCRamplification,treatthemixturewithDpnItoreducebackground.Follow the DpnI manufacturer’s procedure for the reaction and include a DpnI heat-inactivation step before using the DNA mixture in the assembly reaction.

PCR Amplification Werecommendusingahigh-fidelitypolymerase,suchasPhusion® DNA Polymerase, andreducingthenumberofPCRcyclesusedduringamplificationtominimizetheintroduction of errors.Note:ThefollowingprocedureissuitableforPCRamplificationusingPhusion® DNA Polymerase. If you are using a different DNA polymerase, adjust conditions accordingly.

Reaction Set up Component Volume (μL)

InsertorVectorDNA(100pg/μL–1ng/μLinTE) 0.5μL10μMFwdprimer 2.5μL10μMRevprimer 2.5μL10mMdNTPs 1μL5x Phusion HF Buffer 10μLPhusion®DNAPolymerase(2U/μL) 0.5μLNuclease-free Water 33μLTotal Volume 50 μL

Amplification Conditions Initial Denaturation 98°C 30 seconds 1 CycleAmplification

98°C 10 seconds25–30 CyclesPrimer Tm* (60°C to 70°C) 20 seconds

72°C 30 seconds per kb

Final Extension 72°C 5 minutes 1 CycleHold 4°C — 1 Cycle

*You may need to optimize the temperature used for annealing during PCR. If you observelowyield,repeatPCRamplificationwithalowerannealingtemperatureand/orincreasethenumberofamplificationcycles.Ifyouobservenonspecificproducts,repeatthePCRamplificationusingahigherannealingtemperature.

Assembly procedures and additional product information may be found at sgidna.com.