Use for barrier pipet tips

D. KIM dkim at
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.

Daniel Kim

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