Frequently Asked Questions
What is Primordium’s full plasmid and long PCR sequencing?
Primordium sequencing is a new type of overnight DNA sequencing intended to replace Sanger sequencing for routine plasmid cloning.
We use proprietary technology with the goal of providing the following benefits over traditional sequencing:
- No requirement for ordering primers,
- Whole-plasmid and long-PCR sequencing, as compared to ~750 bp traditional reads,
- No issues with GC-rich or repetitive DNA,
- Lower sample mass requirements,
- Faster turn-around time,
- Plasmid purity data
- Automated analysis of errors in plasmid assembly.
How do I submit my samples? How much DNA do I need?
See sample submission guidelines on the ordering page.
For DNA greater than 200 ng/μl, pipette at least 4 μl of your sample into
strip tubes, label the caps as shown
place into a bag with the submission form, and place in the dropbox. For
lower concentration DNA or PCR products, pipette 10 μl into strip tubes.
The details, including dropbox locations and mailing instructions, are available when you submit the samples.
What format do you use to report the data? How do I download my data?
We return your results in three formats: GenBank (.gbk), FASTQ (.fastq), and ABI (.ab1), once the run is complete. After you log in, go to Order and scroll toward the bottom to find your order history. You will see a download button when your results are ready. You will be notified via email when the results are ready for download.
For those unfamiliar with genbank format: This format contains the primary sequence and feature annotations automatically created by alignment to Addgene's publically available feature database.
Sometimes the base calling isn't perfectly accurate or the sample contains multiple contradictory sequences. For this reason we report the quality of the base information at each position using two different formats: FASTQ and ABI.
For those unfamiliar with the FASTQ format: FASTQ is a sequence format (FASTA) with additional quality information and can be read by most modern sequence viewers. Since each base only has one quality value, certain sequence features, such as insertions or deletions, must be inferred from looking at adjacent bases.
The ABI format is popular due to widespread use in Sanger sequencing reporting. This format is intended to depict the traces measured by a Sanger sequencing machine as your sequencing sample passes through the detector. For this reason this antiquated format has a maximum size limit and therefore we report sequences in multiple pieces. Despite some disadvantages, the advantage of the ABI format is that you can get a better sense of the polymorphism present in the sample, and putative insertions can be reported more correctly.
Examples of quality reporting with FASTQ and ABI format are depicted below.
Are there plasmid size limits?
We routinely sequence plasmids up to 25kb without issue, and can sequence up to 40kb plasmids if the DNA is high quality. Generally, larger plasmids benefit from higher concentration DNA.
Can I submit GC-rich samples or samples with repetitive DNA?
Yes, our sequencing technique is accurate for GC rich regions, such as the pCAG promoter sequence, and for repetitive
regions (not including homopolymers), such as artificial promoters.
Why does the gel show a band at twice the expected plasmid size?
Sometimes a virtual gel shows a second (or third) band that is a multiple of the smallest band. These reflect true multimers present in your plasmid prep. They usually go unnoticed, but are quite common, sometimes approaching the concentration of the monomer by mass. They are more common when plasmids have large repetitive regions or other complex structures, and are also more common when grown in RecA+ cells, such as NEB Turbo cells.
Why is the returned DNA sequence only half as big as the band on the gel?
Because the plasmids can form dimers or multimers, we only return the sequence of the monomer. Even if the monomer isn't present as high concentration in the sample, we still return the monomer. You can determine whether your plasmid is mostly monomer, dimer, or multimer by looking at the virtual gel.
What is ZeroPrep sequencing?
ZeroPrep sequencing provides DNA sequences from bacterial colonies, so you only need to prep pre-verified clones. The cost is $20 per colony.
Many colleagues have asked for help with clone screening - nobody wants to waste their time miniprepping a bunch of plasmids to screen for the correct ones.
With ZeroPrep, you send us bacteria and we isolate the plasmid and perform amplification before sequencing it.
Then you can prep only the clones with your desired sequence. Please note:
- ZeroPrep is only available for high-copy plasmids, less than 15 kb.
- We do not amplify plasmids before sequencing, so we require sufficient amount of sample
- Preferred: Pick a large colony and resuspend liquid culture in 10ul of water. The sample should be obviously cloudy, the cloudier the better. These can be processed the day received and yield overnight results.
- Alternative: Provide us with E. coli spotted on an agar plate. It's easiest to streak a 5 mm spot from each colony onto the plate, and then submit the plate. We will incubate the plate overnight at 37C before processing if the colonies are too small or not visible.
- Alternative Two: Provide us with 100 ul culture. We will grow overnight at 37C if the culture isn't dense.
- We do not perform reruns on ZeroPrep samples
- If you are submitting a plate, Onmitrays work best, but any plate will work. Just remember to label the spots and parafilm the plate.
What is Premium PCR sequencing?
Premium PCR has some advantages over standard PCR sequencing, but it takes longer and is more expensive to run:
- Premium PCR sequences from the end of each linear DNA fragment to give individual reads spanning the entire PCR product. Standard PCR starts sequencing from the middle of the fragment, yielding incomplete reads.
- Premium PCR also uses more accurate chemistry, which gives higher confidence per base.
- All reads are returned with Premium PCR, whereas only reads that map to the consensus are available with Standard PCR. There is a low level of multiplexing error, which means that a few reads from your sample might be returned to another customer on the same sequencing run.
- Premium PCR takes about one week to process, is $30/sample, and will typically yield about 3000 reads.
Sample preparation instructions:
- PCR product must be purified over a column (e.g. Zymo C & C) or via magnetic beads (e.g. Ampure XP). Unpurified samples may fail and/or you may be charged a purification fee.
- Submit 5 ul of DNA in EB or water using the chart below. You can submit less concentrated DNA, but your yield might be lower. DO NOT SUBMIT AT HIGHER CONCENTRATION WITHOUT FIRST CONTACTING US.
|Amplicon Size ||DNA Concentration|
What is long PCR sequencing?
We provide sequencing of long PCR products. We accept both purified and unpurified PCR products over 550 bp in length, so long as there is a single band when analyzed on a gel. The first and last ~20 bp will not be accurate.
What limitations are there?
You will not get good quality data if your DNA is degraded or contains contaminants.
A single sequence will be returned if multiple different plasmids are present in your tube (such as a deletion product).
We also cannot currently provide confident reads for homopolymers longer than 8 base pairs, although we are working to be less sensitive to homopolymers.
Here is an example of the failure to accurately resolve the length of the long polyT tract encoding the polyA tail used in the Pfizer/BNT COVID vaccine.
Do you offer discounts?
Yes, for most sample types, we provide a 10 percent volume discount for orders with 96 samples.
Can I submit samples in a 96-well plate?
Although we recommend using strip tubes, we do accept full
96-well plate submissions. Please follow these guidelines:
- Indicate the plate orientation by adding "by row" in the order name if your samples are numbered sequentially by row
(i.e., row A contains samples 1 through 12). Otherwise, we will assume default column numbering (i.e., column 1 contains samples 1 through 8).
- To help our technicians more easily identify the plate orientation, please label the first column (i.e., samples 1 to 8) if you are using column-numbering. Similarly, please label
the firist row (i.e., samples 1 to 12) if you are using row numbering.
- Using sticky film to seal your plates may lead to cross-well sample contamination during transport. A heat sealer works
better. Or, you can dehydrate the plates as described below.
- To prevent cross contamination of samples, our customers have had good success with dehydrating plates:
spin the plate(s) down and then place the plate(s) on a 65 degree heating block for 0.5 to 1 hour or until dry. After dry, you can seal
the plate with sticky film to prevent contamination of the sample during transport. We will rehydrate the sample in our lab for sequencing.
If you want to submit partial plates
, we require at least 48 samples (again strip tubes are preferred). As noted above, if you are numbering your samples
by row (i.e., row A contains samples 1 through 12), please add "by row" to the order name. Otherwise, we will assume default column numbering
(i.e., column 1 contains samples 1 through 8). Please fill up each column/row before moving onto the next column/row.
How can I pay for the service?
You can pay with a credit card/P-card, or pay with an institutional PO (purchase order). A downloadable quote is available
What is the error rate?
We see fewer than one error every 50,000 bp (not counting homopolymers), and any miscalled bases are typically annotated as having low confidence. Our team is heavily focused on continuous improvement, so please let us know if you believe we have returned an incorrect sequence.
I have a suggestion about how you can improve this service. Who can I contact?
Our goal is to accelerate progress in biological research and we welcome any suggestions about how to improve our service. Please contact
firstname.lastname@example.org with any suggestions or comments.
Do you accept international orders?
Yes we accept international orders. Please contact us so we can discuss payment options. We have international shipping notes here
How do I submit mail in samples?
Samples must be submitted in 200 μl strip tubes. Feel free to cut off the unused tubes to use next time. Please make sure caps are closed
securely and place strip tubes inside a container such as a 50 ml conical tube for protection.
If you are using rush shipping or guaranteed delivery options with your courier, please select a delivery time after 9:30 AM.
Our receiving facility opens after 9:30 AM.
Please do NOT use a regular envelope meant for paper letters. Otherwise, the postal service sorting machines will destroy your samples when it goes
through the rollers. We will get an envelope full of crushed plastic pieces and we cannot put that on our sequencing machines.
We recommend shipping samples in a padded envelope via any express courier.
People generally use FedEx, but UPS, DHL, or USPS are all fine.
You can also send the samples in a padded envelope via first class mail.
Multiple orders can be mailed in the same package, so long as each order is in an individual bag with the sample sheet.
Please make sure caps are tightly closed and place strip tubes inside a 50 ml conical tube for protection.
We will notify you if any samples were lost during transport.
Send your samples to:
4743 Poplar Level Rd
Louisville, KY 40213
For our international colleagues, please see our shipping notes here.