Whole Plasmid Sample Prep

• Pick a colony into 1-5 mL of LB with appropriate antibiotic selection like you would for a standard miniprep. Grow at 37°C with shaking at 200-300 RPM.
• The culture should have OD600 > 0.3 for submission, which should look slightly cloudy in a standard 1-5 mL shaker culture. Liquid cultures with OD600 < 0.1 show high failure rates due to insufficient biomass. 
• Send a 20 - 100 μL aliquot of turbid media in 200 μL PCR strip tubes. 
• We do not return samples, so make sure to save the remaining culture for archiving and further expansion.

Tips for best results

• Saturated cultures grown for 12-16 hours show consistently successful sequencing results. If you are short on time, a colony picked from a warm agar plate incubated overnight and inoculated into a 1 mL culture with shaking (300 RPM) will achieve an OD600 > 0.3 within six hours.
• Please do not centrifuge and resuspend saturated overnight cultures in a smaller volume. While it may seem that more biomass is better, this overloads our plasmid extraction chemistry and can result in failed sequencing runs.

This is the fastest method to submit samples and retain a copy of the colony for your own archival or scale up use.

• Pick a colony with a diameter greater than 2 mm into either 20 μL of LB media in 200 μL PCR strip tubes or 96-well microtitre plates. Samples resuspended in 10 μL of water are accepted but not recommended — this submission has higher failure rates and is ineligible for reruns.
• Try to pick the entire colony off of the plate and ensure the entire colony is fully resuspended. We find that picking colonies with a p20 pipette tip and then pipetting up and down three times with the pipette set to 10 μL works well.
• The resuspended colony liquid should appear cloudy upon submission. Insufficient biomass is the most common cause of failed ZeroPrep sequencing runs. 
• Maintain a liquid media aliquot or patch plate copy of your bacteria since we do not return samples.
  • You can generate an archival agar plate by using the same pipette tip you used to pick and resuspend the colony to inoculate a small patch of agar.
  • Alternatively, once candidate colonies are resuspended in the PCR strip tubes, a p20 8-channel multichannel pipette can be used to remove ~0.5 µL of liquid and then spot on to an agar plate in rows. One standard 10 cm diameter petri dish can easily hold four rows of eight spots.
  • Many customers start 1-5 mL miniprep cultures from the resuspended colonies and then only purify plasmids of interest when they receive their sequencing results.
• Send us the remaining resuspension in 200 µL strip tubes.

• For patch plates, spot ~1 cm² patches from individual colonies. Number the patches (Sample#1 in your order is Patch #1). Incubate the patch plate until a small bacterial lawn is visible before submitting.
• For primary transformations or streak-outs of glycerol stocks, circle colonies of interest and assign each a unique number (Sample#1 in your order is Circled Colony #1). Ensure that each colony is at least 2 mm in diameter and that each identified colony is well separated from other colonies upon submission.
• For low-copy plasmids, larger diameter colonies are recommended.
• Please seal the plate with parafilm since the lids will otherwise fall off during transport and place the sealed plate in a ziplock bag. 
• Making a copy of the plate is recommended to ensure you retain material for further processing once you receive your plasmid sequencing data. We do not return samples.
   

200 μL PCR strip tubes (less than 48 samples)

• Set up your order online and receive your unique 6-character order ID, which is the code displayed above the printable QR code sheet.
• Number the tubes. The number can be placed on the side of the tube (preferred) or the cap.
• Write the first 2 characters of your order ID on the 1st tube of each strip.
  

200 μL 96 well plates (48 samples or more)

• IMPORTANT!  For ZeroPrep processing we can only accept samples organized in columns. Label the first column (i.e., samples 1 to 8). Do not leave any empty wells between samples.
• 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.
• Label the front edge of the plate with the full 6-character order ID.

• Set up your order online and receive your unique 6-character order ID.
• Write the 6-character order ID on the bottom of the plate (the part that holds the agar, not the lid!)

• Ensure caps fit snugly and close them firmly. Do not use brands of strip tubes where the caps can easily pop open during shipping. We find that strip-cap tubes tend to open more readily than brands with individual attached caps.
• Ensure caps fit snugly and close them firmly. Do not use brands of strip tubes where the caps can easily pop open during shipping.
• For dropbox submissions, place the PCR strip tubes in a 50 mL conical bottom tube. 
• Print your sample submission form, fold the paper so the QR code is visible, and place the tube and submission form in a ziplock bag.
• If you are mailing in the samples, place the PCR strip tubes in a 50 mL conical bottom tube and then place inside a small box, or bubble wrap shipping envelope) to protect them during shipping. Why? Include your sample submission form with the QR in the package. 

• 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.
• If using our Dropbox service, place each plate in a unique Ziplock bag. Print your sample submission form, fold the paper so the QR code is visible, and place in the same bag.
• If you are mailing in 96-well plates, wrap each plate individually in bubble wrap or other packaging material to prevent cracking and seal punctures. Include your sample submission form with the QR in the package. 
• Please note, don't stack multiple plates directly on top of each other, as the wells of one plate will likely puncture the seal of the next plate.
   

• Seal the plate closed with parafilm and place into a ziploc bag.
• If you are mailing in your sample and not using a dropbox, we recommend protecting the plates with some bubble wrap.

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

1. 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.
    
2. 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.
       
3. 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.

Why do I need these forms? Read more here.