Bacterial Colonies

Please submit your bacterial clones  in a 96-well agar stab plate (one grown colony per well).

Alternatively, you can also submit bacterial clones on an agar plate wrapped in parafilm: 

• Spacing – Please make sure colonies are widely-spaced on your petri dish.  Please allow some room for colony growth during shipping to minimize the chance for plate over-growth.
• Labeling – Circle and label colonies you need sequenced. If you are submitting more than one petri dish, label each with the corresponding order tracking number. When submitting multiple petri dishes for the same tracking number, please make sure that the sample name on the submission form clearly corresponds to the labeled petri dishes (e.g. PlateA-Colony1).
• Logistics – Send colonies in a 96-well or on a petri dish at room temperature in a box with padding. You can also use your Azenta Dropbox. Please contact us for locations and pickup times. Caution: We do not receive samples on Saturdays. Please contact Azenta customer service at DNASeqUK@genewiz.com +44 (0) 1279 873 852 (UK) or sanger.europe@genewiz.com +49 341 520 122-41 (Europe) before sending samples on a Friday.

Submit E. coli containing plasmids. Only small circular templates (i.e. plasmids) can be used successfully with rolling circle amplification. Bacterial chromosomes are not efficiently amplified by the process and cannot be directly sequenced with our Sanger service.

For best results, AVOID the following:

• Cells containing low-copy plasmids. These plasmids may not provide sufficient input DNA for efficient amplification. Using high-copy vectors is recommended.
• EndA+ strains. Certain strains of E. coli (e.g. BL21, Stbl3) contain a non-specific endonuclease in the periplasmic space that can cleave plasmid DNA when cells are lysed, leading to inefficient amplification. Strains with the endA mutation (e.g. DH5α, TOP10) are recommended.
   

Tip: The best way to prevent cross-contamination is by growing colonies in a grid pattern on a petri dish. This is also helpful when clones need to be tracked for use in downstream applications, such as Azenta plasmid preparation service. Simply draw a grid on the back of the plate with alcohol-resistant, indelible ink.

We also sequence colonies that are randomly distributed on a petri dish. Please let us know if positive clones will be used in downstream applications, like GENWIZ plasmid preparation service, after they have been sequenced. 
 

Glycerol Stocks

Please submit 10 – 50 µl of glycerol stock per clone.

• Preparation – Prepare bacterial cultures with 8-30% glycerol in 96-well plates.
• Shipping – Contact customer service on DNASeqUK@genewiz.com +44 (0) 1279 873 852 (UK) or sanger.europe@genewiz.com +49 341 520 122-41 (Europe) for more information.

Submit E. coli containing plasmids. Only small circular templates (i.e. plasmids) can be used successfully with rolling circle amplification. Bacterial chromosomes are not efficiently amplified by the process and cannot be directly sequenced with our Sanger service.

For best results, AVOID the following:

• Cells containing low-copy plasmids. These plasmids may not provide sufficient input DNA for efficient amplification. Using high-copy vectors is recommended.
• EndA+ strains. Certain strains of E. coli (e.g. BL21, Stbl3) contain a non-specific endonuclease in the periplasmic space that can cleave plasmid DNA when cells are lysed, leading to inefficient amplification. Strains with the endA mutation (e.g. DH5α, TOP10) are recommended.
• Rich media. Certain media (e.g. TB, SOB, 2YT) contain high salt concentration or ingredients that can inhibit polymerase activity during rolling circle amplification. LB is recommended.
  
  
  

  VIRAL PLASMID CONTAINING INVERTED TERMINAL REPEAT (ITR)

   

  Be aware that sequencing ITR is challenging, so we request concentrated stocks of DNA.

  
  

  • Optimal sequencing concentration for Viral Plasmid is 300ng / ul (minimum 200ng / ul). Please submit as much template as you are able up to 10ul.
  • Please include exact concentration in Notes field for each template.
  • Ensure 260/230 ratio of 1.8-2.2, as this protocol is particularly sensitive to organic contamination.
  • Primer – Please provide 5 µl per reaction at 5 pmol / µl (5 µM) in a labeled 1.5 ml tube.
  • Generate primers that bind between 150bps and 350bp from ITR.
  • Service does not require barcodes, do not label templates or primers with barcodes.
  • For additional shipping supplies, please contact logistics.europe@genewiz.com.  For UK customers please contact DNASeqUK@genewiz.com.

   

  Note: Turn Around Time for sequencing Viral Plasmid with ITR is 5-7 business days