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VIRTUAL CLONING

Perform virtual cloning using Gateway, Gateway Multisite, Gibson, GeneArt, In-Fusion, TA, TOPO, and PCR-directed restriction enzyme cloning methods. PRICING DOWNLOAD FREE TRIAL

Use SeqBuilder Pro to be confident in your cloning experiment in the lab, every time!

At its core, the process of molecular cloning is quite simple, but without proper planning and preparation, even the simplest cloning experiment will go wrong quickly. Virtual cloning using SeqBuilder Pro enables you to easily and confidently plan your cloning experiment so that you get it right in the lab. SeqBuilder Pro offers unmatched flexibility to help you meet your experiment requirements, including the ability to batch clone groups of fragments simultaneously, and guides you step-by-step in preparing your vectors and amplifying your inserts using all of the most popular cloning methods:  Gateway, Gateway Multisite, Gibson, GeneArt, In-Fusion, TA, TOPO, and PCR-directed restriction enzyme cloning. In silico cloning in SeqBuilder Pro gives you the proper planning and preparation you need to be confident in your cloning experiments, every time!

Perform virtual cloning in 4 simple steps

Virtual Cloning Step 1

Step 1

Select your insert(s) and cloning method

Virtual Cloning Step 2

Step 2

Choose your vector(s)

Virtual Cloning Step 3

Step 3

Check preview, then click Make it

Virtual Cloning Step 4

Step 4

View and analyze results!

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Learn more about Virtual Cloning

Resources | Tutorials | FAQs | Citations | User Guide

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Resources

Please see our resources below for more information on virtual cloning.

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Learn SeqBuilder Pro Workflows

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Structure-guided molecular cloning for improving site-directed mutagenesis and stability in protein design

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Master Our Foundation Application, SeqBuilder Pro

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SeqBuilder Pro FAQ: Answers to Your Webinar Questions

Read Blog Post

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Creating the curated feature library for accurate vector auto-annotation

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Enhanced Sequence Analysis for Today’s Molecular Biologist

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Lasergene Molecular Biology Overview

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Tutorials

Watch one of our videos or check out one of our written tutorials to learn more about using automated virtual cloning in SeqBuilder Pro.

Virtual Cloning in SeqBuilder Pro

This overview shows how to do virtual cloning in SeqBuilder Pro, using Gibson cloning as an example. However, you can perform any of the following cloning methods using the same procedure, by simply choosing the name from a drop-down menu: Gateway, GeneArt, restriction enzyme, In-Fusion, TA, directional TOPO.

Multisite Gateway Pro Cloning in SeqBuilder Pro

Learn how to perform virtual MultiSite Gateway cloning in SeqBuilder Pro.

Automated Clone Verification in SeqBuilder Pro

SeqBuilder Pro can automatically verify whether a lab-made clone that has been Sanger sequenced matches the virtual clone you designed earlier using SeqBuilder Pro. This brief video shows just how fast and easy it is to use this new automated clone verification workflow.

FAQs

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.

Can I clone more than one fragment at a time?

Yes. SeqBuilder Pro offers batch cloning, which allows you to automatically clone one or more fragments into one or more vectors simultaneously. You can choose vectors from the catalog we provide, or use a custom vector. Following batch cloning, SeqBuilder Pro will generate a primer list for each insert/vector combination.

Citations

In vitro studies identify a low replication phenotype for hepatitis B virus genotype H generally associated with occult HBV and less severe liver disease
Vitina Sozzi, Fang Shen, Jieliang Chen, Danni Colledge, Kathy Jackson, Stephen Locarnini, Zhenghong Yuan, Peter A. Revill. Virology, Volume 519, 2018, Pages 190-196, ISSN 0042-6822, https://doi.org/10.1016/j.virol.2018.04.015.

Endogenous melatonin promotes rhythmic recruitment of neutrophils toward an injury in zebrafish
Ren, D., Ji, C., Wang, X. et al. Sci Rep 7, 4696 (2017) doi:10.1038/s41598-017-05074-w.

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis
Pontes-Quero S, Heredia L, Casquero-García V, et al. Cell. 2017;170(4):800–814.e18. doi:10.1016/j.cell.2017.07.031.

In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes
Vitina Sozzi, Renae Walsh, Margaret Littlejohn, Danni Colledge, Kathy Jackson, Nadia Warner, Lilly Yuen, Stephen A. Locarnini, Peter A. Revill. Journal of Virology Oct 2016, 90 (22) 10054-10064; DOI: 10.1128/JVI.01293-16.

Systematic evasion of the restriction-modification barrier in bacteria
Christopher D. Johnston, Sean Cotton, Susan R. Rittling, Jacqueline R. Starr, Gary Borisy, Floyd E. Dewhirst, Katherine P. Lemon
bioRxiv 387985; doi: https://doi.org/10.1101/387985.

Expression of Omp16 and L7/L12 Brucella abortus protective antigens as secretory fusion proteins in mammalian cells
Prusty, Bikash R, Tabassum, Rizwana, Chaudhuri, Pallab, Saini, Mohini, Chaturvedi, V K, Mishra, B P, Gupta, Praveen K. Indian Journal of Biotechnology. Vol 16, July 2017, pp 289-295.

Non-canonical Helitrons in Fusarium oxysporum
Chellapan, B.V., van Dam, P., Rep, M. et al. Mobile DNA 7, 27 (2016) doi:10.1186/s13100-016-0083-7

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    Andrea Todd, NRC- Saskatoon

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