Bacteria, Phage, BAC DNA, and AAV-ITR

Choose the custom service type when sequencing from a bacterial colony, glycerol stock, phage with circular genome (supernatant or plaque), or BAC DNA. For these services, please add one additional day to the turnaround time.

For each reaction, provide 5 µl of your primer at 5 pmol/µl (5 µM) in a labeled 1.5 or 0.5 ml tube.

Bacterial Colonies

Please submit bacterial clones on an agar plate wrapped in parafilm.

• Spacing – Please make sure colonies are widely-spaced on your agar plate.  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 plate, label each with the corresponding order tracking number. When submitting multiple plates for the same tracking number, please make sure that the sample name on the submission form clearly corresponds to the labeled plates (e.g. PlateA-Colony1).
• Shipping – Send colonies on an agar plate using Standard Overnight 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 Technical Support at 1-877-436-3949 option 2 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 an agar plate. This is also helpful when clones need to be tracked for use in downstream applications, such as plasmid preparation. Simply draw a grid on the back of the plate with alcohol-resistant, indelible ink.

We also sequence colonies that are randomly distributed on an agar plate. Please let us know if positive clones will be used in downstream applications, like plasmid preparation, after they have been sequenced. You can do this by selecting the “Save for Prep” checkbox at the top section of the DNA sequencing order form.

Glycerol Stocks

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

• Preparation – Prepare bacterial cultures with 8-30% glycerol in 1.5 ml or 2 ml microcentrifuge tubes. For sequencing many samples, use 96-well plates.
• Shipping (US) – Glycerol stock samples must be sent on dry ice. Please do not send glycerol stock samples on Fridays because we do not receive samples on Saturdays. Please do not use your Azenta Dropbox for any dry ice packages.

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.

Phage with Circular Genomes (US Services Only)

Please submit 50 µl of phage supernatant in 8-strip PCR tubes or 1.5 ml microcentrifuge tubes, or provide phage plaques in a petri dish.

• Titer – Please indicate the titer in the “Comments” section of the order form.
• Large Projects – Please contact us to set up a pilot study. This usually consists of 3-4 dilutions to determine the optimal sequencing conditions.

Bacterial Artificial Chromosomes (BAC DNA)

Be aware that sequencing BAC DNA is challenging, so we request concentrated stocks of DNA and primer.

• BAC DNA – For each sequencing reaction, provide 2.5 µg or more in 15 µl. The BAC DNA concentration should be at least 150 ng/µl.
• Primer – Please provide 5 µl per reaction at 10 pmol/µl (10 µM) in a labeled 1.5 or 0.5 ml tube.
• Large Projects – Please contact us to set up a pilot study.

Note: Sequencing BAC DNA requires an additional day of turnaround time.

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 ration 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 or 0.5 ml tube.
• Generate primers that bind between 150bps and 350bp from ITR