Primer Walking Frequently Asked Questions

1. What is primer walking?

This technique, also known as “directed sequencing” utilizes a series of Sanger sequencing reactions to either confirm the reference sequence of a known plasmid or PCR product based on the reference sequence (sequence confirmation service), or to determine the unknown sequence (sequence discovery service) of a full plasmid or PCR product by designing primers to sequence overlapping sections.

At GENEWIZ, we offer full-service primer walking workflows. Just send your samples and reference sequence, if available. Our team will synthesize all the necessary primers, perform the Sanger sequencing, analyze the raw data, and deliver the full consensus sequence of your plasmid or region of interest along with the raw data.

2. What is the application of primer walking?

GENEWIZ’s primer walking service is commonly used for determining the full sequence of a plasmid/construct prior to downstream use. It has been useful to researchers who want to verify their newly constructed plasmids via Sanger sequencing, or to determine the full sequence of a construct where very little information is known.

3. What is the difference between the "sequence confirmation" and "sequence discovery" service options?

If a reference sequence for the plasmid is available, our team will perform "sequence confirmation" which includes analysis of the sequencing data compared to the reference sequence. In case of an unknown plasmid sequence, our team will perform "sequence discovery" by designing primers using a known piece of the sequence, such as, information from the vector backbone, antibiotic resistance gene, specific promoter, etc., and then use generated Sanger sequence to design the subsequent primers.

4. What is the turnaround time for GENEWIZ’s primer walking services?

The average turnaround time for sequence confirmation is 3-5 business days once the primers have been created. In order to expedite your projects, please provide your reference sequence or primer requests immediately, so we can begin designing the assay before your samples arrive.

The average turnaround time for sequence discovery is 1 week per 2bk of template as primer design depends on previous sequencing.

5. What if I do not know my complete reference sequence?

Even if some information about the plasmid is known, our team can design the first primer from that information and “walk” the plasmid. An antibiotic resistance gene or another known region of the backbone vector are some examples of the information pieces that our GENEWIZ team can utilize.

6. What is the difference between Sanger sequencing and primer walking?

Primer walking is a full-service option that utilizes Sanger sequencing to sequence your entire plasmid or PCR product. For primer walking, we design and synthesize the primers necessary to sequence your target region or whole plasmid. We also, analyze the Sanger sequencing data and match it to the reference sequence or create a plasmid map.

GENEWIZ’s standalone Sanger sequencing service accepts plasmids or PCR products as starting material to perform Sanger sequencing. For this service you can either submit your own primers, use GENEWIZ universal primers, or order primers from GENEWIZ. We return raw sequence data only.

7. What type of starting material does GENEWIZ accept?

GENEWIZ accepts the following sample types for both the "sequence confirmation" and "sequence discovery" service options: plasmid DNA, bacterial colonies on agar plates, glycerol stocks, agar stabs, and purified or unpurified PCR products.

We recommend starting with total RNA. However, we have extensive experience performing extractions from a wide variety of materials, including cell pellets, fresh frozen tissue, blood, and FFPE slides. We also accept sorted cells for ultra-low input RNA-seq. Check out our Sample Submission Guidelines for more information.

8. What DNA concentration does GENEWIZ require for primer walking?

Plasmid – Submit 1 µg per kb of the target sequence for single-stranded coverage, 2 µg per kb sequence for double-stranded coverage, and 4 µg per kb for 4x coverage. Submit at least 10 µg plasmid if region of interest is of unknown length (for sequence discovery). Please submit a minimum concentration of 200 ng/µl per sample.

 PCR Products – Submit 10-40 ng per kb of amplicon.

 ITRs – For plasmids and PCR products that contain ITRs, please submit an additional 1 µg of material per ITR in the sample sequence.

 Primers – We can synthesize all the primers if the known reference sequence is provided (for sequence confirmation). We can synthesize the initial primer if the sequence for a known region (e.g. antibiotic resistance) is provided (for sequence discovery). If you have primers that can be used in the initial round of sequencing, please submit a minimum of 10 µl each at 5 µM.

 Packaging – For DNA and primers, please use 1.5 ml screw cap tubes or microcentrifuge tubes secured with Parafilm and label them with the sample or primer name and concentration.

9. What if I don’t have enough DNA to submit?

If your sample does not meet the minimum DNA concentration required for optimum primer walking per GENEWIZ standards, you can place an order for GENEWIZ’s plasmid DNA preparation services either through your GENEWIZ account or by working with one of our project managers. Our primer walking team can then use this newly generated DNA as a starting material for your primer walking order.

10. Do you accept PCR products?

Yes, GENEWIZ accepts either purified or unpurified PCR products.

11. What coverage options does GENEWIZ offer?

GENEWIZ’s primer walking service offers 1x, 2x, and 4x coverage for both "sequence confirmation" and "sequence discovery" orders.

12. What type of data can I expect?

GENEWIZ provides raw sequencing data in .ab1 and .seq formats, and consensus sequence data in .seq and .fasta formats.

13. What does a primer walking report contain?

For "sequence confirmation", the primer walking report contains a constructed consensus sequence per sample along with any observed variants compared to the reference sequence.

For "sequence discovery", the primer walking report contains a map of the plasmid or region on interest.

Have a specific question?

Email | Phone 1 (877) 436-3949 ext. 3350

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