To miniprep or not to miniprep, that is the question.

To sequence a large batch of clones, we typically pick clones into 96 well plates containing 50-80 ul of LB/amp with 7.5% glycerol. The plates are grown for 2-3 hours at 37C (can be done in mini-orbital shaker).

At this point, you can use this to innoculate a deep well block for O/N culture and miniprepping. This is the preferred method if your clones contain repetative DNA such as an enriched CA library. Miniprepping gives the best quality DNA for sequencing with no repetative stutter. If you want to sequence through the repetative DNA, and maximize your chance of getting the sequence all in one reaction (just the forward without having to do the reverse), it is best to miniprep and then sequence.

For non-repetative DNA you have more choices. Since O/N cultures requires waiting overnight to prep things and since miniprepping can give uneven yields due to variations in clone growth (requiring DNA quantification), it is not always optimal. Miniprepping also costs about $1 per well. An alternative is to PCR the clone inserts, clean up the PCR product (using SPRI with magnetic beads) and then directly sequence these products. In some ways, this is cheaper as the beads are quite inexpensive. I have used this method very successfully for non-repetative DNA. It especially makes sense when you want to PCR for insert size anyway so you are already running the PCR reactions.

There is a new method by Hawkins (Elkin et al. Genome Research 11: 1269) which needs to be tried. This method uses magnetic beads directly on cell culture without any pelleting and resuspension.