1. Option 1: Liquid Culture
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Liquid cultures are a convenient option for customers who already grow their clones prior to sequencing.
- Inoculate a colony into 1–5 mL of LB media containing the appropriate selection antibiotic.
- Grow the culture at 37°C with shaking at approximately 200–300 RPM.
- Aim for an OD600 greater than 0.3 before submission. The culture should be noticeably cloudy.
- Transfer 20–100 µL of the turbid culture into a 200 µL PCR strip tube for sequencing.
Cultures with very low cell density can contain insufficient bacterial biomass and may be more likely to produce unsuccessful results.
Since submitted material is not returned, retain the remaining culture for storage, additional growth, or downstream experiments.
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2. Option 2: Cell Pellet
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Grow your bacterial culture under the appropriate conditions for your strain and construct.
Transfer 1–5 mL of culture to a suitable centrifuge tube and centrifuge until a compact cell pellet forms. Carefully remove the supernatant without disturbing the pellet and remove as much residual liquid as possible.
Do not resuspend the pellet. Securely close and label the tube, then submit the cell pellet directly for EasyPrep and ONT sequencing.
What to submit: A cell pellet prepared from 1–5 mL of bacterial culture, with the supernatant removed.
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3. Option 3: Colony
Select this option if you have not grown up your colony and need us to grow it OR if you are submitting a resuspended colony.
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Colony
You can submit colonies directly on an agar plate, either as individual colonies or as labeled colony patches.
For colony patches:
For colony patches, create approximately 1 cm² patches, with each patch originating from an individual colony. Clearly number each patch so that it corresponds with the sample number in your sequencing order.
Allow the plate to incubate until visible bacterial growth has developed before submission.
For individual colonies:
Clearly circle and number each colony you would like sequenced. Colonies should be:
- At least 2 mm in diameter
- Well isolated from neighboring colonies
- Clearly labeled to match the corresponding sample in your order
For low-copy plasmids, selecting larger colonies is recommended.
Before shipping, secure the plate lid with Parafilm and place the sealed plate inside a protective resealable bag. Do not use too much parafilm or it can cause issues when unwrapping.
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Colony - Resuspended
Colony resuspension provides a quick way to submit samples without preparing an overnight culture or performing a plasmid extraction.
- Select a well-isolated colony measuring at least 2 mm in diameter.
- Transfer the colony into 20 µL of LB media in a 200 µL PCR strip tube or compatible 96-well plate.
- 10 µL of water may also be used, but LB media is preferred because water-based submissions may have a greater risk of insufficient sample input.
- Transfer as much of the colony as possible and mix thoroughly until the cells are evenly suspended.
- The finished suspension should appear visibly cloudy, indicating sufficient bacterial material.
- Submit the remaining suspension in the appropriate PCR strip tube or plate.
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