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.
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)
* 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.