Post-reaction Clean Up & Resuspension

This protocol works on either 1/8th or 1/16th scale reactions in a 96-well reaction plate.*

Centrifuge speeds are given in both RCF and RPM. The Bay Paul Beckman TJ-25 centrifuge should be set with RCF units.
Steps 4 - 9 should be carried out without delay to prevent loss of pellets.

DNA in formamide will degrade even if kept frozen. Reactions should be precipitated within 24 hours. After resuspension they should be sequenced within 3-5 days. If longer times elapse your sequences might be somewhat shorter. If long reads are a priority, be sure to contact JBPC to schedule your sequencing before you set up reactions.

Supplies: Isopropanol 75 % and 70%, pipette tips, reservoir tray, paper towels, Costar foil sealer, HiDi formamide**

Devices: centrifuge with microtiter plate carriers, pipette (8 or 12-channel preferred)

  1. Spin plate briefly.

  2. Add 30µl 75% Isopropanol, seal with Costar foil, invert a few times to mix, shake sample to bottoms of wells.

  3. Incubate at room temp for 15 minutes.

  4. Centrifuge at 2800 RCF or 4200 RPM for 30 minutes.

  5. Remove foil, invert on paper towel, "swirl" inverted plate and paper towel in circular motion on benchtop until most IPA falls out.
    Do not bang/slap/slam/tap inverted plate or pellet could be lost. (Alternatively a short spin at 100-200 RCF can be used. It is not necessary to get every last bit of IPA out of the wells.)

  6. Add 50µl 70% IPA, seal with foil.

  7. Spin at 2000 RCF or 3300 RPM for 10 minutes.

  8. Remove foil and invert onto fresh paper towels.

  9. With plate inverted on paper towel, spin at 200 RCF or 500 RPM for 1 minute.

  10. Air dry for 20 minutes in the dark.

  11. Resuspend in 7µl HiDi Formamide.
    (Note: Add formamide to all wells, even if you do not have a full plate of samples, or you will cause damage to the 3730XL capillary array.)

  12. Spin plate briefly.

  13. Seal with Costar foil. Store in freezer and plan to sequence within 3 days.

* If you wish to use a 384 well plate, substitute 14 µl of 86% IPA and 10 ul of 70% IPA in steps 2 and 6, respectively.

** Formamide is a relatively inexpensive part of the sequencing process. Use only ABI's HiDi formamide. It should be stored at -20ºC with minimal freeze/thaw cycles. Aliquot into 1.5ml tubes to reduce freeze/thaws, and if it does not freeze when stored at -20ºC do not use it.

Last updated 11/05/06