Summary

Sample Preparation

Concentration

Incorrect concentration is the leading cause of failures. Kindly submit your standard, substantial, or oversized plasmid samples adhering to the specified concentration and minimum volume provided in the table above. Quantify the samples using a Qubit or an equivalent fluorometric method, such as a plate reader. We do not recommend using Nanodrop for quantification because it is not reliable enough for the sequencing equipment.

Meticulous and precise quantification, followed by proper normalization, is paramount for successful sequencing. Over or under concentrating samples will most likely have an adverse impact on the results.

Quality

For optimal outcomes, strive for the presence of pristine, circular double-stranded plasmids. Plasmids that exhibit degradation or fragmentation pose a higher risk of sequencing failure, as they may fail to yield a consensus due to a lack of complete sequencing reads.

To verify size, conduct assessments on full-length plasmids (not digested or amplified) through gel electrophoresis. For linearized plasmids, employ a linear ladder, while intact circular plasmids benefit from a supercoiled ladder. It's essential to note that Sanger sequencing and PCR amplification are insufficient for size verification since these methods rely on primers to detect specific small regions.

The workflows for significant plasmids (25 - 125 kb) and extensive plasmids (125 - 300 kb) exhibit greater resilience to degradation, given the inherent difficulty in extracting plasmids of these larger sizes without some degree of degradation. Nonetheless, to ensure optimal results, the objective remains intact circular DNA even within these more forgiving workflows.

The linear/amplicon workflow exhibits greater resilience to degradation compared to the plasmid workflow, owing to its incorporation of minimal fragmentation during the library preparation process. Nevertheless, to achieve optimal outcomes, the pursuit of intact linear DNA molecules remains a paramount consideration.

Purity

The cleaner the product, the better the results. Leftover material in the sample can act as an inhibitor during sequencing. We suggest opting for samples exhibiting a 260/280 ratio exceeding 1.8 and a 260/230 ratio falling within the range of 2.0-2.2. Purity assessments can be conducted using Nanodrop or spectrophotometric methods, although these methods cannot be used to accurately measure the concentration.

For optimal outcomes, it is imperative that samples do not include any of the following components:

• Denaturants, such as guanidinium salts, phenol, etc., or detergents like SDS, Triton-X100, etc.
• Leftover contaminants from the organism.
• RNA (we recommend RNase treatment during extraction for its removal)
• Any insoluble material that would cause colors or cloudiness.

Furthermore, samples should contain copies of a single clonal molecule. You can send mixtures but it may yield inconsistent results. Submit mixtures at your own risk.

Sample Preparation

Sample preparation for Linear / Amplicon samples is the same as whole plasmid samples. Refer to the WPS section for details.

Sample Preparation

Bacterial Genomic DNA (gDNA) Samples

To facilitate the seamless execution of this service, it is imperative that customers provide 1 µg of high-quality, high-purity, and high-molecular-weight (HMW) double-stranded genomic DNA (gDNA). The optimal specifications include having more than 50% of the DNA exceeding 15 kb in length, with a recommended purity ratio of 260/280 surpassing 1.8 and the 260/230 ratio falling within the range of 2.0-2.2.

Notably, our affordable pricing and rapid turnaround times do not encompass quality control (QC) services for incoming samples. Hence, it becomes the submitter’s obligation to ensure that the prepared samples adhere to these stipulated requirements before shipping. This proactive verification step is essential to guarantee the successful processing of your bacterial gDNA samples.

Sample prep steps

1. Prepare sample from a bacterial clonal culture
   • There are various methodologies available online in the public domain for how best to prepare bacterial cultures. We encourage researchers to seek out the protocols that fit their needs.
2. Extract and purify sample from clonal culture
   • Again there are many different extraction methods available on the market. Eurofins Genomics is indifferent to the type of extraction method as long as it produces high-quality, high-purity, high-molecular-weight (HMW), double-stranded genomic DNA (gDNA) devoid of nicks, gaps, breaks, and contaminants is deemed suitable for this sequencing service. Here are a few recommendations for extraction kits from trusted brands:
   • Zymo
     • Zymo® Quick-DNA Miniprep Plus Kit
     • Zymo® Quick-DNA Fungal/Bacteria Miniprep Kit.
   • Wizard - Wizard® Genomic DNA Purification kit
   • Qiagen
     • Qiagen® MagAttract HMW kit
     • Qiagen® DNeasy kit
   • Additional Tips
     • Refrain from vortexing.
     • Use wide-bore tips for pipetting.
     • Elute in elution buffer instead of water.
     • Avoid exposure to high temperatures (>37°C) for more than 1 hour, extreme pH levels (<6 or >9), intercalating fluorescent dyes, or UV radiation.
     • Steer clear of freeze-thaw cycles; store gDNA at 4°C for 1-2 months.
     • If utilizing a speed-vac, avoid heat and be careful not to over-dry.

3. QC the sample before shipping it

This step involves 3 areas: quantity, quality, and purity.

Quantity: It is imperative to provide 1 µg of genomic DNA (gDNA) at a concentration of 50 ng/µL in 20 µL of elution buffer. We recommend using a Qubit for quantification or another fluorometric method, such as a plate reader, and we discourage the use of Nanodrop.

For high molecular weight (HMW) gDNA, additional homogenization efforts, such as an extended incubation time, elevated incubation temperature, and thorough, gentle mixing, may be necessary for precise quantification. Adequate homogeneity is typically indicated when separate DNA quantifications from the top and bottom of the sample differ by less than 15%.

If <1 µg was obtained from the first extraction, we highly recommend performing additional extractions to meet the yield criteria. You can submit <1 µg but at your own risk. If submitting <1 µg, prepare the sample at the required concentration (50 ng/µL) but in a reduced volume based on your total yield. Also, an email to GenomicsSupport@eurofins.com prior to shipping and always appreciated so we know what to expect.

Quality: over 50% of the total DNA should be above 15 kb in size. If not, we highly recommend extracting and purifying again. There are multiple options for size characterization, including Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder.

Purity: the minimum purity for gDNA samples is 260/280 ratio above 1.8 and a 260/230 ratio between 2.0-2.2. Acceptable options for testing purity include Nanodrop or other spectrophotometric methods. If the sample does not meet the recommended criteria, please re-extract or cleanup using a Qiagen cleanup kit or AMPure XP beads.

Additional Tips

- No RNA. The best way to prevent RNA is to use an RNase treatment during extraction.
- No denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.).
- No residual contaminants from the organism/tissue (heme, humic acid, polyphenols, etc.).
- No insoluble material or exhibit coloration or cloudiness.

Cell Pellets for the Bacterial DNA Extraction Option

Cell pellets from BSL1 strains are allowed. BSL2 is not allowed. Pellets should be reconstituted in Zymo 1X DNA/RNA Shield. Furthermore, we encourage customers to cultivate a freshly grown clonal culture of your bacteria in liquid broth. The best time to harvest cells is doing the growth stage or early stationary phase. Sending cells from older cultures in the death phase is discouraged.

Please note that Eurofins Genomics does not cultivate samples. Our lab will extract DNA from the material you submit. Without adequate cell collection from the culture, the extraction process is likely to fail. Lastly, this service is only available in the US due to customs regulations.

Sample Prep Steps

1. Prepare the cell pellets
   1. Centrifuge the cells into pellets to remove excess supernatant. A compact cell pellet should weight approximately 15 mg and not exceed 50 mg. The requisite quantity is equivalent to 8-12 OD600 or 4-6 x 10^9 cells (e.g., 8-12 mL culture at 1.0 OD600).
      1. In the case of wet cell pellets (e.g., Streptococcus sp.), where complete removal of supernatant without disturbing the pellet is not feasible, an approximate weight of 30-50 mg is recommended.
   2. Resuspend in 1 mL PBS, followed by another round of centrifugation to remove further supernatants.
   3. Conclude the process by resuspending the pellet in 0.5 mL of Zymo 1X DNA/RNA Shield inside a 2 mL tube.

2. Submit order
   1. Go to the order page and select the correct size. .
   2. Be sure to print the confirmation page and folder it inside the tube bag when submitting/shipping the samples. Ensure that each tube is clearly labeled with the order code, and sample number.

3. Ship sample
   1. There are multiple options for how to submit samples, including dropboxes, digital shipping labels (provided free during checkout for all orders >$30), or ship using your own shipping carrier.
   2. To safeguard against potential damage and leakage during transportation, it is essential to position the screw cap tubes within a sturdy container, such as a falcon tube or tube box, before dispatching. For orders exceeding 10 samples, specifically arrange the tubes within a tube box, loading the samples row by row in numerical order. This meticulous organization significantly streamlines the sample reception process, reducing handling time. Please dispatch the samples at room temperature for optimal conditions.

Sample Requirements
To ensure the best sequencing results, please follow the guidelines below when submitting your Premium PCR samples:

• Sample Type: Linear, double-stranded DNA only (single-stranded DNA may result in unpredictable results).
• DNA Purity: DNA should be purified using a column-based method (e.g., Zymo Clean & Concentrator) or magnetic beads (e.g., Ampure XP). Unpurified DNA will not be accepted.
• Volume: A minimum of 10 μL per sample is required, submitted in 200 μL strip tubes.
• Buffer: Use elution buffer (10 mM Tris, pH 8.5) or nuclease-free water. Avoid using buffers containing DMSO.
• Concentration: For optimal sequencing results, send your samples at the following concentrations based on the size of your DNA insert:
• 100 bp: 1 ng/μL
• 200 bp: 2 ng/μL
• 500 bp: 5 ng/μL
• 1000 bp: 10 ng/μL
• 2000 bp: 20 ng/μL
• 5000 bp: 50 ng/μL
• 10000 bp: 100 ng/μL
• 15000 bp: 150 ng/μL
• 20000 bp: 200 ng/μL
• 25000 bp: 250 ng/μL

Verify Concentration

Please provide your Premium PCR samples at the recommended concentration and minimum volume listed in the table above. For optimal sequencing performance, we highly recommend measuring DNA concentration using a Qubit or another fluorometric method (such as a plate reader), as these provide the most reliable results.

If you choose to use spectrophotometric methods like Nanodrop, keep in mind that these tend to be less precise for DNA quantification. Samples submitted at concentrations that are too high or too low can compromise library preparation and sequencing, potentially leading to unsuccessful runs. In fact, incorrect sample concentration—often caused by reliance on Nanodrop readings—is the most frequent reason for sequencing failure. Careful quantification and proper normalization are essential for consistent, high-quality results.

Verify Quality

This service is designed specifically for linear, double-stranded DNA. We suggest confirming the size of your full-length DNA molecules using gel electrophoresis before submission.

Verity Purity

We recommend that DNA samples have a 260/280 ratio above 1.8 and a 260/230 ratio between 2.0 and 2.2. Purity can be assessed using Nanodrop or other spectrophotometric methods.

To ensure optimal sequencing performance, samples should be free of:

• RNA (treat with RNase during extraction if needed)
• Denaturing agents (such as guanidinium salts or phenol) and detergents (like SDS or Triton-X100)
• Residual biological contaminants from the source organism (e.g., heme, humic acids, polyphenols)
• Insoluble particles, visible coloration, or cloudiness

This service is primer-free, so please don’t send primers—separately or mixed in.

We rely on you to prepare samples that are clean and ready for sequencing. We do not provide DNA extraction or QC services. Use this checklist before shipping:

Concentration:
Normalize your samples to the required volume and concentration, measured using Qubit or a comparable fluorometric method. Do not use Nanodrop for concentration—it’s unreliable for this purpose.

Quantity:
DNA must be double-stranded. Verify the full-length size using gel electrophoresis:

• Use linear ladders for linear DNA
• Use supercoiled ladders for circular DNA
• For DNA >10–15 kb, we recommend pulsed-field electrophoresis with high-molecular-weight markers

Purity:
Ideal absorbance ratios:

• 260/280 > 1.8
• 260/230 between 2.0–2.2
  Nanodrop can be used only for purity checks—not for quantification.

Samples must be free of:

• RNA (RNase treatment strongly advised)
• Denaturants (e.g., guanidinium, phenol)
• Detergents (e.g., SDS, Triton-X100)
• Biological contaminants (e.g., heme, humic acid, polyphenols)
• Insoluble material or visible cloudiness/color