Use for barrier pipet tips
dkim at nmsu.edu
Tue Jun 25 12:11:04 EST 1996
I recently contributed to the discussion of agarose gel freeze and
squeeze, suggesting that a barrier tip may be used as a "fritted column"
for centrifugal removal of agarose from DNA solution.
Here is another suggestion. I have tried it and it works (most of the
time). This is a procedure for doing tiny plasmid minipreps in a 96-well
plate. One drawback of microplate minipreps is the need for a microplate
centrifuge bucket . . . an uncommon and expensive item. I have minimized
centrifugation steps and modified the final lysate clearing step to
reduce the amount of individual tube "hands on" time.
1. Grow bacteria in wells of a 96-well microplate (I use open-top
polyethylene microwell plates, which are not necessarily sold as sterile,
but are good enough :)). Use 100 microliters of medium plus antibiotics
(two drops from a 10-ml pipet). Cover the plate with Saran wrap,
carefully squeezing air from the wrap to make it cling to the rims of the
wells. shake overnight.
2. Next day, 50 - 80 microliters remain of the cultures. Add 5 - 10
microliters of TE with lysozyme and RNAse A (you can figure out the
concentrations). shake for a few minutes.
3. Add 1 drop (about 50 microliters) of NaOH/SDS. Shake a few
minutes. The lysates should clear.
4. Add 1 drop KOAc solution and shake a few minutes. Lysates should
form a ppt.
5. Array 96 1.5 ml tubes in racks. Add to each tube a barrier tip.
6. With a truncated yellow tip, transfer all the material from each
well into the top of the tips. Spin briefly (5 - 10 s) in a microfuge
(yes, the tubes are open).
7. Discard the tips. The cleared lysates should be about 100 microliters.
8. Add 1 vol isopropanol and cap and shake. Spin out the DNA 10 minutes.
9. Put all the tubes back into a rack, caps open. Cover the whole
array with a piece of glass and invert over a sink. The alcohol should
trickle out of the tubes.
10. Bring the tubes upright and wash gently by adding a bit of 70%
ethanol to each tube from a squirt bottle. Cover with a glass plate and
drain again. Repeat (remember to wash the caps of the tubes).
11. Invert a final time and let the tubes sit on the glass plate
until the alcohol has dried out.
12. Dissolve in 10 microliters RE digest mixture and incubate, etc.
I don't know why anyone would regularly need to do hundreds of minipreps
in a day, but if you do, here is a way to keep your sanity.
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