Sample Preparation and Shipping

Bacterial Genome Sample Prep

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Bacterial Genome Sequencing with Extraction

Service Level	Size	Sequencing Platform	# of Cells	Preservative	Cost
Standard	<7 Mb	ONT	4-6 x 10^9	500 µL Zymo 1X DNA/RNA Shield™	$105
Big	7-12 Mb	ONT			$120
Hybrid	<7 Mb	ONT + Illumina			$180
Big Hybrid	7-12 Mb	ONT + Illumina			$210

Count your cells.

We require 4-6 x 10^9 cells (usually about 8-12 mL of culture at OD600 of 1.0). We accept cell pellets from both BSL1 and BSL2 strains.

Wash and pellet cells.

Pellet the cells by centrifugation and remove as much supernatant as possible.

Resuspend the pellet in 1 mL PBS to wash, then pellet again and remove supernatant.

Tips for best results:

A compact cell pellet with all the supernatant removed (e.g. E. coli pellets) should weigh approximately 15 mg.
A wet cell pellet where supernatant cannot be all removed without disturbing the pellet (e.g. Streptococcus sp. pellets) should be approximately 30-50 mg.
Please don’t send more than 50 mg of material or there will be too many cells to be protected from degradation by the Zymo 1X DNA/RNA Shield™.

Resuspend the pellet.

Resuspend the final pellet in 500 µL of Zymo 1X DNA/RNA Shield™ in a 2 mL screw cap tube (when ordering Zymo DNA/RNA Shield™, you can enter PLASMIDSAURUS at checkout for 10% off).

Please do not use any other type of preservation media, as they do not preserve the DNA as effectively and do not fully inactivate the host cells, which is required for safe handling in our lab.

Label your tubes.

Set up your order online and receive your unique 6-character order ID.

Label each 2 mL screw cap tube with the first two digits of your order code, sample number, and a plus (+) or minus (-) to indicate whether each strain is gram positive or gram negative. For example, "XX3-" would indicate order code XXYYZZ, sample 3, gram negative.

Package your tubes.

Ensure caps are closed firmly.

Place the labeled tubes into a small protective vessel (such as a Falcon tube, a small box, or bubble wrap) to protect them during shipping (Why?). If using a box, please load the samples row by row in numerical order, as this greatly reduces the handling time when receiving your samples.

Send cell pellets at room temperature. Dry ice and cold packs are not necessary.

Dropoff or ship.

Print out your order form. Fold so that QR code is readily visible and place in a small bag with your samples.

For fastest results and easiest shipping, place your samples in a Plasmidsaurus Dropbox!

If you ship your samples to us directly:

Place packaged samples in a sturdy cardboard or padded envelope or a cardboard box. If reusing old packaging, ensure that any hazard warnings are completely obliterated. Multiple orders can be shipped in the same package, as long as each order is placed into a separate bag with its corresponding order confirmation sheet.
Do not add ice packs or dry ice. Samples should be shipped at room temperature.
Ship via any Express or First-Class shipping carrier such as FedEx, UPS, DHL, or USPS. The shipping address will be provided on your order confirmation sheet.

If you aren't using a Plasmidsaurus dropbox, print and fill out the following documents:

Commercial Shipping Invoice - Bacteria

For best results, make sure to include the shipper's identity, shipper's address, and the quantity of items within the shipment.
Detailed Description - Bacteria
1. If your samples do NOT include animal derived additives then use the template for NO animal derived additives and provide a detailed name or description of the microbe/recombinant product, including genetic insert. Add your institution's letterhead, the date, and a signature.
2. If they DO include animal derived additives then use the template for WITH animal derived additives. Provide a detailed name or description of the microbe/recombinant product, including genetic insert, as well as a list of the animal derived additives. Add your institution's letterhead, the date, and a signature.
Toxic Substances Control Act (TSCA) Certificate - Bacteria (only if shipping from outside the US to US)

Further guidance for international shipping

The information below constitutes only a recommendation for shipping samples classified as "non-regulated materials" to our facility. Due to continuing changes in state and federal regulations, clients should always check with their safety office and/or shipping department to ensure regulatory compliance.
Make sure all forms are fully completed. Incomplete and/or missing forms will likely cause delays and other issues.
Keep an eye out for any communication or information requests from your shipping company of choice in case of issues. It is your responsibility to provide any information they request, and we advise following their guidance as their systems will have the most up-to-date customs information.

Exporting into the USA

Samples must be packaged and shipped in accordance with U.S. Department of Transportation (DOT) and/or International Air Transport Association (IATA)/ International Civil Aviation Organization (ICAO) and carrier regulations.
Your international courier will require you to complete a commercial invoice to be included with the shipment.
The commercial invoice should describe the contents as “non-hazardous, non-contagious research sample.” Please list your institution as the "Manufacturer" of the samples if Manufacturers Identification (MID) code is required.
State that the “contents are Not Restricted under IATA DGR regulations.”
We recommend that you include a Toxic Substance Control Act (TSCA) Certification. If you do not include this, the package may be delayed at customs until the courier receives the proper certification.
Declare the value of the goods to be $1.
We only accept pure DNA samples that are intended for Research Use Only! We DO NOT accept any samples for clinical diagnostics or trials. Any samples that do not conform to our submission guidelines will be discarded and/or submitted to the United States Postal Inspection Service

Exporting into Germany

If shipping to Cologne, certain sample types will require some additional information. Please reach out to support if you have any questions.
Category B infectious substances (UN 3373) are classified as being potentially harmful to humans or animals and must be packed in accordance with ADR (International carriage of dangerous goods by road) packing instruction PI 650 for road transport and IATA packing instruction PI 650 for air transport.
Unlike other dangerous goods, UN3373 shipments do not require a Shipper's Declaration. However, the Air Waybill (AWB) must include: “UN3373, Biological Substance, Category B” Net quantity
UN3373 (Biological Substance, Category B) requires triple packaging per Packing Instruction 650 (PI650) to ensure safe transport. This consists of a leakproof primary receptacle, a leakproof secondary packaging with absorbent material, and a rigid outer package, with proper labeling (UN3373 diamond).
Shipping UN 3373 Biological Substances (Category B) into Germany requires strict adherence to international (IATA for air, ADR for road) and national regulations to ensure safety and compliance with customs. Please make sure you are following these regulations.

Exporting into Singapore

The commercial invoice should describe the contents as “non-hazardous, non-contagious research sample.” Please list your institution as the "Manufacturer" of the samples if Manufacturers Identification (MID) code is required.
Please make sure to use the correct HTS code to ensure fastest processing. The HTS code we use for our shipments is found on the template commercial invoice document above.
We recommend including a detailed customs description (the template for which can be found above), or a Material Data Safety Sheet (if you have one already available for your samples). This is not required, but can be requested by customs if a delay occurs.

Why do I need these forms? Read more here.

Bacterial Genome Sequencing without Extraction

Service Level	Size	Sequencing Platform	Normalized Concentration	Minimum Volume	Cost
Standard	<7 Mb	ONT	50 ng/μL	20 μL	$90
Big	7-12 Mb	ONT		20 μL	$105
Hybrid	<7 Mb	ONT + Illumina		30 μL	$165
Big Hybrid	7-12 Mb	ONT + Illumina		30 μL	$195

Extract and purify genomic DNA.

Use your favorite method to extract and purify genomic DNA from your clonal bacterial culture. RNase treatment is required during extraction.

Any method that yields high quality, high purity, high molecular weight (HMW), double-stranded gDNA that is free of nicks, gaps, breaks, and contaminants is suitable. Our favorites are the Zymo Quick-DNA™ Fungal/Bacteria Miniprep Kit and the ZymoBIOMICS™ 96 MagBead DNA Kit.

Caution: We strongly recommend against any extraction methods that include phenol and/or chloroform, as these samples always have extremely poor sequencing performance with ONT long reads, even if the QC metrics after gDNA extraction look normal.

Be gentle with your gDNA

Avoid vortexing and fast or unnecessary pipetting; pipet with wide-bore tips only
Elute in elution buffer, not water
Do not expose to high temperatures (>37°C) for >1 hour, pH extremes ( <6 or>9), intercalating fluorescent dyes, or UV radiation
Avoid freeze-thaw cycles; store gDNA at 4°C for 1-2 months
If using a speed-vac, avoid over-drying of gDNA; do not use heat in the speed-vac

Samples should NOT contain any of the following:

Sequencing primers
RNA (RNase treatment is required during extraction)
Denaturants (guanidinium salts, phenol, etc.) or detergents (SDS, Triton-X100, etc.)
Residual contaminants from the organism (heme, humic acid, polyphenols, etc.)
Insoluble material, colors, or cloudiness

Perform quality control checks.

Our low prices and fast turnaround times do not include gDNA quality control (QC) services, so it’s important that you verify that your samples meet our requirements prior to shipping them to us.

Measure gDNA concentration with fluorometry

We require performing DNA quantification assay with Qubit or equivalent fluorometric method such as a plate reader. Spectrophotometric methods like Nanodrop are much less accurate and submitting the wrong concentration can cause sequencing failure!
- Please note: High molecular weight (HMW) gDNA often requires extra homogenization effort (longer incubation time, increased incubation temperature, very extensive gentle mixing, etc.) to obtain accurate quantification. If separate DNA quantifications from the top and bottom of the sample are within 15% of each other, this is usually a good indication of adequate homogeneity.

Measure gDNA purity with spectrophotometry

This step is strongly recommended. We recommend gDNA samples with 260/280 of ~1.8 and 260/230 between 2.0-2.2. Purity assay may be performed with Nanodrop or other spectrophotometric methods. If your sample is contaminated and does not meet this metric, either re-extract the sample or clean up the sample to remove the contaminants using a Qiagen cleanup kit or AMPure XP beads.

Assess gDNA quality with gel electrophoresis

This step is strongly recommended. Get the most out of long-read sequencing by keeping your fragments long. We recommend gDNA samples with >50% of the total DNA mass contained within fragments above 15 kb in size. If the sample does not meet this metric, we strongly recommend repeating extraction. Size characterization may be performed with Femto Pulse, Fragment Analyzer, Bioanalyzer, or a slab gel with a HMW ladder (ideally with pulsed-field electrophoresis).

Normalize genomic DNA concentration.

Submit your final purified genomic DNA normalized to 50 ng/μL in the minimum volume of elution buffer (10 mM Tris, pH 8.5) that is specified in the table above.

If you don’t have enough DNA to reach the minimum volume at 50 ng/μL, we’d recommend extracting more DNA if possible. If that’s not possible, send us an email at support@plasmidsaurus.com and submit a lower volume at 50 ng/μL.

Label your tubes or plates.

Set up your order online and receive your unique 6-character order ID.
Write the first 2 characters of your order ID on the 1st tube of each strip

Set up your order online and receive your unique 6-character order ID. On the order page, please check the “Is this a plate submission?” checkbox, then select if your plate is numbered sequentially by column or by row.
Label the front edge of the plate with the full 6-character order ID.
If you are using column numbering, label the first column (i.e., samples 1 to 8). If you are using row numbering, label the first row (i.e., samples 1 to 12).

Load and package your sample.

Add the correct volume of your sample(s) from the table above. Make sure to seal and package your samples well to make sure they arrive safely for sequencing!

Ensure caps fit snugly and close them firmly. Do not use brands of strip tubes where the caps can easily pop open during shipping.
Place the labeled tubes into a small protective vessel (such as a Falcon tube, a small box, or bubble wrap) to protect them during shipping. Why?

Start with well A1 and load your samples contiguously. Our preference is for samples to be loaded by column but we can accept samples loaded by row if indicated in the order. Do not leave any empty wells between samples.
Ensure your plates are sealed extremely thoroughly to survive shipping without leaking — sadly we receive a lot of leaky plates. We recommend using a heat seal (rather than adhesive) or snugly-fitting strip caps on top of the plate. If you only have adhesive options, click here for some examples of sealing technique.
For RNA-Seq samples in Zymo DNA/RNA Shield, do not use adhesive seals, as the adhesive will dissolve and the wells will leak. We strongly recommend only using strip caps.
Wrap each plate individually in bubble wrap or other packaging material to prevent cracking and seal punctures. DO NOT stack multiple plates directly on top of each other, as the wells of one plate will likely puncture the seal of the next plate.