96-well Plasmid DNA Purifications

The automation group has developed a high quality DNA purification system in a 96-well format.

Prior to Submission

  • We recommend a 96-well, round bottom 2.0ml block (preferred vendor: USA Scientific, cat. 7556-9600, 2.0 ml deep 96-well polypropylene TiterBlock®, square wells, natural, 20/pack).The volume of media per well should be 1.5ml.
  • Please label the front of your block with the automation order number. Label theBside of the plate so the label faces you when well A1 is located in the top left-hand corner.
  • Growth conditions are important. Overnight growth of deep-well blocks in incubators equipped with shaking mechanisms that can properly aerate the cultures as well as the use of a gas-permeable seal (preferred vendor: Worldwide Medical Products, cat. 41061023, BioexcellAeraseal, 100/pk) is ideal. If you are using a richer media, such as Terrific Broth (TB) or Yeast/Tryptone (2XYT), please know that your cultures will grow faster than those cultures grown in Luria-Bertani (LB) broth and therefore reach stationary phase sooner. Doing an optical density read time-course experiment will help in determining your proper incubation period.Our purification protocol requires that the OD600 of 1.5ml of culture be no greater than 3.0 prior to plasmid preparation. We suggest an optimal OD600 of 2.5-2.8.
  • Grown cultures should be pelleted by spinning at >3000RPM for approximately 15 minutes. Please gently tap the block when decanting to remove all media. Plates should be sealed with an aluminum seal (preferred vendor: Worldwide Medical Products, cat. 41061019, AlumaSeal II, 100/box) and deposited at your designated drop-off location.
  • If you wish to have your samples sequenced at our facility, please select 'Plasmid Prep plus Sequencing' when placing a Plasmid Preparation order. Be aware that in accordance with our new sequencing submission guidelines, plate position H12 MUST be left empty. Please include a tube containing 500µl of sequencing primer (at a concentration of 10ng/µl), and label the primer tube with the automation order number.Note: If your sequence contains G-C rich regions or secondary structure, it is highly recommended that you order the "Difficult Template" sequencing protocol to ensure successful sequencing.

Results

  • DNA will be eluted in 50µl/well Elution Buffer (5mM Tris, pH 8.5) into a v-bottom polystyrene plate and sealed. Eluted DNA will be delivered to your desired MGH drop-off location or may be picked up at the facility.
  • OD260 readings will be kept on file and are available upon request.