Please fill each well of a 96-well, round-bottom 2.0 ml block with 1.5 ml of Terrific Broth (TB).
You can purchase deep 96-well blocks from USA Scientific (2.0 ml deep 96-well polypropylene TiterBlock®, square wells, natural, 20/pack; Cat # 1896-2000).
If you wish to have your samples sequenced at our facility, well H12 MUST be left empty for an internal quality control!
Inoculate each medium-containing well with a single colony.
Seal the deep-well plate with a gas-permeable film.
We recommend to purchase gas-permeable seals from Worldwide Medical Products, Inc. (BioExcell Film for Tissue Culture, 100/pk; Cat # 41061023).
Incubate the plate at 37°C in a shaking incubator to ensure proper aeration of the growing cultures.
Allow inoculated cultures to grow to an optical density (OD600) of 2.5-2.8.
The use of a rich medium such as TB requires the optimization of the growth time. Our 96-well Plasmid Preparation protocol is optimized for use with bacterial cultures grown to an OD600 value of 2.5-2.8. Please make sure that the OD600 is NOT above 3.0!
Once the cultures have reached an OD600 of 2.5-2.8, collect the cells by centrifuging the growth plate at 3000 rpm for approximately 15 minutes. Immediately decant the medium, turn the block upside down and gently tap it on a paper towel.
Seal the block containing the bacterial cell pellets with an aluminum seal.
We recommend to use aluminum seals from Worldwide Medical Products, Inc. (Aluma Seal II, 100/bx; Cat # 41061019).
Place your 96-well Plasmid Preparation order online. Please label the front of
your growth block as clearly as possible with the Automation order number
(label faces you when well A1 is located in the top left-hand corner).
IMPORTANT: 96-Well Plasmid DNA Preparation PLUS Sanger DNA Sequencing
If you wish to have your purified Plasmid DNAs sequenced at our facility, please enter a combined Plasmid Prep plus Sanger DNA Sequencing order.
Our Sanger DNA Sequencing Submission Guidelines require plate well H12 to be left empty for an internal process control.
Please include a separate tube containing 400 µl of the appropriate sequencing primer at a concentration of 1.5 µM (1.5 pmole /µl), labeled with the Automation order number. If you are planning to submit multiple sequencing primers with your plate of harvested cells, please arrange your primer set in a separate 96-well PCR plate (at least 10 ul of a 10 ng/ul (1.6 uM) primer solution). Well positions of primers must match the well positions of your plasmid clones.
If your DNA templates should contain GC-rich regions, hairpin structures, or repeats (di- and trinucleotide stretches), we highly recommend that you order the Difficult Template Sequencing option to ensure successful sequencing.
Include a printed copy of the barcoded order form with your sample submission.
Please submit your samples, including a hard copy of the completed order form, by using one of our remote dropboxes (please see here) or by delivering it personally to our core facility.
If remote or direct sample drop-off should not be an option for you, samples may be shipped on dry ice to the core facility, using a courier delivery service of your choice (for example, overnight service of FEDEX, UPS, etc.). Please include a hard copy of the completed order form.
IMPORTANT: Please note that we are not receiving samples on Saturdays and Sundays or on official holidays.