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CLONE SEQUENCE VERIFICATION

Confirm the accuracy of your clone and inspect regions of disagreement. PRICING DOWNLOAD FREE TRIAL

Use SeqBuilder Pro to confirm the accuracy of your clone created in the lab.

You’ve finished creating your clone in the lab and are ready to move on to downstream experiments, but how do you know that all your steps have resulted in the expected recombinant clones? Many researchers verify their clone accuracy through Sanger sequencing. We make it easy to perform clone sequence verification to confirm that your Sanger sequencing results perfectly match the clone you designed. Our clone sequence verification workflow confirms that the clone fully contains the insert of interest in the correct orientation, is in-frame with the vector, and confirms that there is adequate coverage of trace files across the entire clone. If discrepancies are identified during the clone sequence verification process, we show you exactly where the disagreements lie, so that you can address them. Use SeqBuilder Pro’s clone sequence verification workflow to catch read misalignment and insertion errors before proceeding with downstream experiments.

Perform clone sequence verification in 4 simple steps

Clone Sequence Verification Step 1

Step 1

Create a virtual clone

Clone Sequence Verification Step 2

Step 2

Choose Cloning > Verify Clone

Clone Sequence Verification Step 3

Step 3

Add sequencing reads, then click Run

Clone Sequence Verification Step 4

Step 4

Interpret results

Learn more about Clone Sequence Verification

Resources | Tutorials | FAQs | Citations | User Guide

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Resources

Please see our resources below for more information on clone sequence verification.

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Learn SeqBuilder Pro Workflows

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Master Our Foundation Application, SeqBuilder Pro

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Cloning and Primer Design Workflows Webinar

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SeqBuilder Pro FAQ: Answers to Your Webinar Questions

Read Blog Post

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Transitioning from SeqMan Pro to SeqMan Ultra

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Introducing Lasergene 17

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Accuracy of de novo assembly of Sanger trace data: SeqMan Pro versus three
alternative pipelines

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Lasergene Molecular Biology Overview

View Document

Tutorials

Watch one of our videos or check out one of our written tutorials to learn more about using clone sequence verification 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.

Sanger Sequence Assembly

See how to assemble and analyze Sanger sequencing data using SeqMan Ultra in this 2-minute video.

FAQs

What virtual cloning methods are supported for clone sequence verification?

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.

What are the essential requirements of the clone sequence so that it can be used for clone sequence verification?

The clone sequence must contain both a vector and insert GenBank-style feature so that SeqBuilder Pro can verify the clone. Any clone created in SeqBuilder Pro will automatically contain these two features.

How do I interpret the clone sequence verification results?

SeqBuilder Pro will produce a Clone Verification Summary that provides a clone schematic that shows where the Sanger/ABI reads aligned, regions of mismatch, and a list of SNPs. If the degree of mismatch is significant, the summary page will clearly state that the clone disagrees with the aligned sequence data.

How do I visualize the aligned Sanger data that verifies my clone?

The SeqBuilder clone sequence verification report contains hyperlinks that will open the alignment in SeqMan Ultra, part of Lasergene Molecular Biology, so that the user can inspect the conflicting regions and SNPs within the context of the alignment.

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

computer Ready to learn more? PRICING DOWNLOAD FREE TRIAL
  • Extremely Valuable

    “SeqBuilder Pro is easy-to-use and intuitive. It is also extremely valuable for complex cloning and mutagenesis projects.”

    Gilbert Martinez, University of Washington

  • Intuitive

    “Easy-to-use, intuitive interface, makes working with sequences easy.”

    Matthew Nilles, University of North Dakota

  • Quick and Easy

    “Quick and easy in silico cloning, RE digestion, PCR primer design and assessment, addition of features.”

    Andrea Todd, NRC- Saskatoon

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