The UW-Madison Biotechnology Center is currently open and following the university’s COVID-19 Response regarding campus operations.
Our plasmid sequencing and assembly service aims to provide you with a complete view of your plasmid and allows for characterization of the target gene and verification of the plasmid backbone. Confirming orientation and number of tandem repeats of the insert and presence of promoters, cut sites, and origin of replication are simultaneously possible. Not only will you receive a FASTA file containing the consensus sequence of your plasmid and an annotated map of your plasmid, you will receive greater assurance that your construct can be used in downstream applications.*
* There is no guarantee of plasmid assembly. Please see our Guidelines and FAQs for how you can have the greatest success with assembly of your plasmid. Where greater nucleotide resolution or independent confirmation is necessary, we offer other services that can help resolve assembly of difficult plasmids!
Plasmid Delivery Vessel
- Strip tube: Must be 0.2ml V-bottom in a strip of 8 consecutive tubes. Fill an entire strip of 8 before proceeding to the next strip. On one side of the strip, label each tube containing a plasmid in consecutive numeric order (e.g. 1-8). On the other side of the strip, add your full name initials to each tube containing a plasmid (e.g. “JL” for Joshua Lederberg). All tubes must have tight-fitting caps. When shipping, please follow additional guidelines below.
- Plate: Must be 0.2ml V-bottom, semi- or full-skirt, 96-well plate. Label the front edge of each plate with your PI (or company) name, initials, “Plate 1”, and today’s date (e.g. Lederberg JL Plate 1 9-16-22). If you are submitting more than one plate, the subsequent plates should be labeled in consecutive numeric order (e.g. Plate 2, Plate 3). Seal each plate with tight-fitting caps or seal. When shipping, please follow additional guidelines below. Submitting in a plate indicates you intend to have 96 plasmids sequenced and assembled. The flat rate pricing applies to all plate submissions.
Plasmid Volume, Concentration, and Size
- 20-30µl at 30ng/µl – All plasmids will be processed as submitted. For volumes <20µl, there is a risk of insufficient volume transfer. While lower or higher concentrations may work, follow the specifications for greatest success.
- <30kb – Larger plasmids are likely to get insufficient coverage for assembly. Please contact us to see how we can help you assemble your large plasmids, fosmids, cosmids, and bacmids!
Requesting Service and Submitting Plasmids
- Submission Form – Complete this form and send it to email@example.com along with how you plan to submit your plasmids.
- Drop-off – There are two locations on campus – one in the Biotech Center hallway outside of room 1360 and the other at WIMR outside room 7140. The Biotech Center drop-off location is monitored for submissions every business day. The WIMR drop-off is monitored for submissions prior to 8am on Mondays, Wednesdays, and Fridays.
- All plasmids can be sent at ambient temperature in a padded shipping envelope or box. Plasmids may be sent on dry ice, but isn’t necessary. We take no responsibility for lost or damaged shipments.
- Strip tubes must have tight-fitting caps and a single layer of parafilm across the caps to prevent them from opening during shipping. Place the strips inside a 50ml conical tube or another hard container to keep from being crushed.
- Plates must be sealed with tight-fitting strip caps and a single layer of parafilm across the caps to prevent them from lifting or sealed with a thermal seal to prevent leaking and cross-contamination. At ambient temperatures, adhesive seals will likely leak. Plates can be sent on dry ice to keep the plasmids frozen and prevent the plate from leaking. Wrap plates in bubble wrap, sandwich between cardboard, or place inside a secondary protective container to avoid cracked wells.
- A physical copy of the submission form must be included in the shipment.
425 Henry Mall Madison WI 53706
* Flat rate pricing is ~$13/sample (UW System) with 96 samples. Flat rate pricing will be charged when plasmids are submitted in a 96-well plate.
Important Notes and FAQs
Does UWBC perform QC of my plasmid DNA? No upfront QC of the input DNA or QC of the final library is performed. We highly recommend that you perform fluorometric quantification (e.g. Qubit) of your plasmid before submitting to ensure you meet the specifications listed in our guidelines.
Do you hold onto my plasmid and can I get it back? Once the data is delivered, we dispose of the plasmid. We suggest you retain sufficient plasmid for your needs as we will not send it back to you.
When can I expect results? If your plasmid is submitted and delivered to us by 8am on Tuesday, the data will be delivered by Thursday of that week. The exception may be weeks with intervening holidays where the facility is closed. If the sample is later than 8am on Tuesday, it will be processed the following week. Please note that shipments received at our loading dock on Tuesday will not reach us in time – please plan shipments accordingly.
What do I get back from UWBC? For each successfully sequenced and assembled plasmid, we will provide a pLannotate map and FASTA file. Soon, we will be providing a custom report with additional details! More information on pLannotate can be found here.
How do I access my data? Our preferred method to distribute data is through Globus. More information can be found here. You will receive an email when your data is available and it will contain instructions on how to access it.
How long do you store my data? We store your data for 60 days. After 60 days, it is deleted from our servers. Please download your data as soon as you receive the data delivery notification!
Do you guarantee success or read counts? Following our guidelines gives the highest chance of successful plasmid assembly. High quality plasmids that meet specifications are very likely to get more than sufficient coverage. We don’t guarantee successful assembly, circularization, or annotation of the plasmid or minimum read count or coverage. If your plasmid failed to assemble, there may be issues other than quality and quantity to consider such as complexity of the insert. This may be addressed with other methods – we’re here to help!
Do you still charge if no data is generated? Each plasmid will be charged, even if no data is generated. Refer to our guidelines for the best chance of a successfully assembled plasmid.
Why do I see indels and frameshifts in my assembly? These are usually associated with homopolymeric nucleotides. The length and frequency of homopolymers increases the occurrence of indels. Even though error correction is performed on the assembled reads, these regions are still prone to single nucleotide indels.
Why should I choose Plasmid Sequencing and Assembly over other methods? This service is great for cost efficient screening without having to design primers. The entire plasmid can be sequenced, assembled, and annotated, providing greater confidence in use of your plasmid in downstream applications!
What type of basecaller is used? Guppy basecaller with the Super accurate (SUP) model is used, which is the most accurate model available for Oxford Nanopore data.
Why do I not see a single, “perfect” peak in the summary report (.html) for read length distribution? Reasons for seeing multiple peaks include overall low sample quality/purity and specifically, plasmid degradation and/or nuclear DNA contamination. Below are some examples of these read length histograms.
Unless otherwise specified, all data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Sequencing Facility are intended for research purposes only. They are not intended nor certified for diagnostic or clinical use. Clinical services are provided through our collaboration with the UW Collaborative Genomics Core.