DNASTAR

What virtual cloning methods are available?

SeqBuilder Pro supports MultiSite Gateway Pro cloning, Gateway cloning, Gibson cloning, In-Fusion cloning, GeneArt cloning, TA cloning, PCR-directed restriction cloning, and directional TOPO cloning.

How do I use restriction enzymes to do in silico molecular cloning?

You can do manual restriction enzyme cloning by directly selecting two enzyme labels and the sequence between them. Use Cloning > Copy Restriction Insert to copy the restriction enzyme. Then use Cloning > Paste Restriction Insert to paste it into the vector. SeqBuilder Pro will check whether the ends are compatible and allow you to modify them, if necessary.

How do I create a TA clone?

TA cloning requires a custom insert and a custom vector, so there are three steps involved:

1) To create a custom insert, select a portion of sequence and use Priming > Create Primer Pairs to create a PCR primer pair. Then use Priming > Create Insert by PCR to extract the sequence of the PCR product and simulate its amplification with Taq polymerase, including the addition of 3’ A overhangs to each end.

2) To create a custom vector, use Cloning > Change Overhangs to open a dialog where you can automatically or manually create the specific type of overhang required for TA cloning. You can then save the new vector to the Cloning Vector Catalog to use in future projects.

3) Create the TA clone using the Cloning > TA Cloning command. Specify the insert created in step 1 and the vector saved in step 2. Press Try It, then Make It.

How do I create a Gibson clone?

After selecting a sequence or range (e.g., a feature on that sequence), use the Cloning > Gibson, LIC Cloning command. Choose a suitable vector from the drop-down menu. Then click the Linearize button and choose cut sites from the Left Cut and Right Cut menus. Press Try It, then Make It to make the Gibson clone. The whole procedure takes under a minute.

Can I use custom vector for Directional TOPO cloning?

Use Cloning > Change Overhangs to open a dialog where you can automatically or manually create the specific type of overhang required for TOPO cloning. The 5’ end must be blunt, without any overhangs, while a specific overhang (gtgg) must be created at the 3’ end of the forward strand. You can then save the new vector to the Cloning Vector Catalog to use in future projects.

Can I add a custom vector to the list I see in SeqBuilder Pro’s Vector Catalog?

Yes. Use File > Open to open the Vector Catalog (CloningVectors.sbp); vectors are organized into folders such as Entry Clones, Donor Vectors, pGEM, etc. Decide which folder is best for your custom vector, then right-click on it and choose Import. Navigate to and select your custom vector, then use File > Save Project to save your vector as part of the Vector Catalog.

What’s the best way to do in silico MultiSite Gateway Pro cloning?

Select Cloning > Multisite Gateway Pro Cloning. Right-click on the Inserts folder, choose Import, and navigate to the insert sequences you wish to use. Once they have been imported, drag the inserts into the Fragments area on the bottom right; then drag and drop them in the desired order. Select a vector from the Vector drop-down menu. Press Try It and then Make It.

Can I validate my clone with sequence data?

Once you have created a clone in the lab and sequenced it, you can validate or verify it using SeqBuilder Pro. Use Cloning > Verify Clone or—during in silico cloning—accept the prompt to verify the clone. Use the Clone drop-down menu to specify the clone and the Set Insert Location button to specify the insert. Use Browse files for Reads and navigate to the Sanger ABI files obtained from sequencing the laboratory clone. Press the Run button to generate a report showing the accuracy and orientation of the insert.

Do any cloning methods support creation of a multiple fragment insert clone?

Any of SeqBuilder Pro’s wizard-guided PCR based cloning methods (Gibson assembly, In-Fusion, GeneArt) and Multi-Site Gateway cloning allow you to easily clone multiple fragments.