Sample Preparation
What types of samples should I prepare for Plasmid-EZ?
Please submit plasmid DNA normalized to a concentration of 50 ng/μL in a minimum volume of 10 μL. Samples should be prepared in microcentrifuge tubes, sealed 96-well plates, and capped PCR strip tubes. If submitting for plasmid DNA preparation plus Plasmid-EZ, please submit at least 15 μL.
We will perform linearization upon receipt of plasmid DNA samples followed by library preparation and sequencing.
Does the starting material need to be normalized to 50 ng/μL?
Yes, as we do not adjust the concentration prior to library preparation. Instead, we use equal volumes for each sample. For optimal results, please adjust your sample concentrations to 50ng/μL prior to submission. We recommend using a dsDNA quantification assay such as QubitTM or PicoGreenTM.
Note: NanoDrop® cannot distinguish between dsDNA and ssDNA (oligos and dNTPs) and may cause you to overestimate the amount of dsDNA in your samples.
How should I prepare the tubes/plates?
Tubes for <48 Samples
If you are submitting <48 samples, please use 8-strip, 0.2 ml PCR tubes and caps. You can cut off empty tubes and use them next time.
GENEWIZ's Online Ordering System will assign tube ID codes using your initials and the sample number (see Figure to the right). Please write these codes on the sides of your tubes using indelible marker.
Label the side of your tubes with YOUR initials and sample number. These should match your order receipt.
Plates for >48 Samples
If you are submitting 48 or more samples, we recommend using a 96-well, semi-skirted PCR plate. Cap the wells with 8-strip caps. These are usually ordered separately from the plates. Be sure that the caps seal tightly! The semi-skirted plate helps to prevent the plate from bending in transit, resulting in fewer loose caps. Please ship the plate with cushioning to avoid potential damage.
Please contact us for help finding a vendor if your laboratory does not have any 96-well PCR plates or matching strip caps.
Arrange your samples vertically (in columns) as shown to the right:
.
What is the size range of plasmids that I can prepare for sequencing?
Plasmids up to 25 kb can be sequenced using this workflow. If you are sequencing larger than 25 kb, please reach out to us for a technical consultation.
Can a mixture of plasmids be submitted for sequencing using this service?
This service is intended for a clonal population of plasmids. If plasmid pools or mixtures are submitted, the sequencing and analysis results cannot be guaranteed.
What happens if my samples do not meet your starting material requirements?
Please submit an inquiry.
Sample Submission
When should I submit samples?
Samples may be submitted at any time. Samples must be received in the morning at our facility to be eligible for that day's cycle. Projects with incomplete information may be placed in the next sequencing cycle. Results are delivered the same day in select regions or in as fast as 1 business day.
What are the sample submission guidelines for Plasmid-EZ?
Sample Type: Purified Plasmid DNA
Size: Up to 25 kb; if larger than 25 kb, please reach out to our NGS experts
Minimum Amount: 500 ng (we recommend using a dsDNA quantification assay such as Qubit or PicoGreen)
Concentration: Normalized to 50 ng/μL
Purity (A260/280): 1.8-2.0
Buffer: Water, EB, or low TE (<0.1 mM EDTA)
How can I send my samples to GENEWIZ?
Please send your samples, along with a copy of your order receipt, to us using one of the following options.
Option #1: Submit samples into a local dropbox at room temperature. We have an extensive network of convenient dropboxes located throughout the US. To find a dropbox near you, please submit an inquiry. Place the samples and order receipt together in a Ziploc bag
Option #2: Ship samples directly to our NJ facility using the shipping address listed on your order receipt. You can ship at room temperature or on blue/dry ice.
What are the cutoff times for my dropbox pickup?
When submitting an order in our customer portal, the final step is to locate a dropbox closest to your institution; please choose the dropbox you will leave your sample at for Plasmid-EZ. During checkout, consult the Order Summary page to find out the daily cutoff times for your selected dropbox (see example below).
Ordering & Processing
How do I order Plasmid-EZ?
You can order Plasmid-EZ in minutes without a quote. Check out the Plasmid-EZ Order Guide for the 9 easy steps.
How much sequencing coverage is expected per plasmid?
Coverage is highly dependent on sample quality as determined by the end-user prior to sample submission and therefore, cannot be guaranteed.
How can I monitor the progress of my Plasmid-EZ project?
The estimated delivery date for your order can be viewed anytime through our customer portal on the Order Summary page. Because of the fast turnaround time of the service, we do not provide detailed project updates (i.e., timestamps for QC, library preparation, sequencing, and data analysis).
What is the turnaround time?
Samples are processed every weekday. Results are available the same day in select regions or in as fast as 1 business day.
Does GENEWIZ perform QC on my Plasmid-EZ samples prior to library preparation?
No, we do not perform sample QC. This enables us to provide rapid and cost-effective service for whole plasmid sequencing. Please measure the concentration of your samples (preferably using a dsDNA quantification assay such as Qubit or PicoGreen) and normalize to 50 ng/μL prior to submission.
Can GENEWIZ perform plasmid DNA preparation for me?
Yes, plasmid preparation can be selected as an add-on service with your Plasmid-EZ order. We will retain your submitted plasmid DNA samples for downstream plasmid preparation. At least 2 μg of plasmid DNA (concentration >100 ng/μL) is required for both services. You can submit bacterial colonies or clonal glycerol stock as starting material for upstream plasmid DNA preparation.
Results
Can Plasmid-EZ detect plasmid multimers?
Sometimes, multiple plasmid copies combine via homologous recombination to produce a multimeric form of plasmid with repeating sequences. This process is called plasmid multimerization. Plasmid-EZ is an effective way to detect such multimers; it generates exceptionally long reads that can detect large repeat sequences within a plasmid. However, if there are multimers that are present at a higher proportion in the sample, they can cause misassembly.
What data output will I receive for Plasmid-EZ?
You will receive an interactive data report, consensus sequence of your plasmid(s) in FASTA format, raw reads in FASTQ files, GenBank files, and annotation files, including plasmid map images.
Additional analysis options are coming soon.
How do I download my data?
Once data analysis is complete, your data will be delivered electronically by sFTP link, email, or through our customer portal. If applicable, login credentials and instructions on how to retrieve your data will be included in the data delivery email sent to you. Please feel free to contact us should you have trouble retrieving your data. You may also refer to the sFTP Data Download Guide for step-by-step instructions and troubleshooting tips.
Will my Plasmid-EZ data be secure?
We take data security very seriously and make every attempt to keep your data private and protected. The data transfer we offer is secure; however, should you desire an alternative delivery method, we are happy to work with you.
Is there additional bioinformatics available?
All custom bioinformatic analysis requests are taken on a case-by-case basis after a technical consultation with one of our next generation sequencing (NGS) study managers. Please note that any customization outside our standard deliverables will incur additional charges and time.
Troubleshooting
What if my plasmid samples fail to sequence?
It is likely that sequencing failure, i.e., failure of the sample to produce consensus sequence with at least 10x coverage or less, may be due to samples not prepared at the required DNA concentration, containing a mixed plasmid population, or degraded/fragmented DNA.
To achieve optimal sequencing results, please follow our recommended sample submission guidelines.
What if my results do not match my expectations or hypothesis?
We cannot guarantee the results of the sequencing data. If you feel that a processing error may have occurred, please contact us and we will investigate on a case-by-case basis.
Direct Colony to Plasmid-EZ
What is Direct Colony to Plasmid-EZ?
Direct Colony to Plasmid-EZ is our whole plasmid sequencing service, starting from bacteria instead of purified plasmid. We will isolate your plasmid preparation prior to sequencing for an added charge.
What types of samples should I prepare for Plasmid-EZ?
- Please submit plasmid DNA normalized to a concentration of 50 ng/μL in a minimum volume of 10μL.
- Samples should be prepared in microcentrifuge tubes, sealed 96-well plates, and capped PCR strip tubes.
- If submitting for plasmid DNA preparation plus Plasmid-EZ, please submit at least 15μL.
- Our team of Plasmid-EZ experts will perform linearization upon receipt of plasmid DNA samples followed by library preparation and sequencing.
- We also accept bacterial samples as starting material. Please visit our sample submission guidelines to learn more.
How do I submit my bacteria?
You can submit your bacteria in several different ways!
- Colonies grown on an agar plate
- Colonies can be selected by circling them on the plate, or we can pick colonies at random
- Colonies picked in liquid media
- Glycerol stock
Note: High-copy plasmids are preferred (less than 15kb). Submit colonies that will grow in 37degC. Any cells that require modified growth conditions may not sequence well.
When will I receive the results?
Data will be delivered within 2 business days after samples are received in our lab.
Will the plasmid preparation used for whole plasmid sequencing be returned?
The plasmid preparation used is an unpure prep and may not be suitable for downstream processes, therefore we do not return from this service.
If you require your prepped plasmid returned, please place a Standard Prep order from the Plasmid DNA Preparation section in your customer account. Then, select Plasmid-EZ verification.
Have a specific question?
Contact Us | Phone 1-877-GENEWIZ (436-3949), ext. 1