Pooling and Purifying
Purpose:
To pool one or more 384-well PCR plates down to one 96-well plate for loading.
Procedure:
We purify into a U.S.A Plastics 96-well plate. One 384- MJ Research PCR plate is purified
into one 96-well plate. We use a robot, the ALH from SiClone, to do this. Pooling action
goes to the upper left quadrant (#1), indirectly. First we (or the robot) dilutes out the 6-FAM
product (blue), as this is the most intense color. Pooling ratio should be about 1:2:2
Fam:Hex:Ned. We add 3ul of water to each well containing blue, then go back and pick up
4ul of each well and pool together into the 96- well plate. By hand, an 8 or 12 channel pipette
works well. Again, one quadrant of four in the 384-well plate gets pooled into one well in a
96-well plate.
Row 1 384-well
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2 Blue PCR mix
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4 Yellow PCR mix
Each quadrant of four is one DNA sample. Well #1 is one marker and well #2 is another, etc…
The same markers are repeated in one row (or more depending on sample number.
Row 1 96-well
A
B
1-4
1-4
C
D
E
F
G
H
1-4
1-4
1-4
1-4
1-4
1-4
One 96-well row, showing the four markers pooled into each well.
After the samples are pooled, add 80ul of 75% iso-propanol. Mix the samples a minimum of
20 times with an up and down action (no bubbles). If you have a greater volume of sample
(i.e. pooling more than 4ul each well), increase the iso-propanol accordingly.
Cover with Micro-Amp 96-plate covers from Perkin-Elmer and place in a dark space at room
temperature for 45 minutes, minimum. The plates can sit longer than that, just not over-night.
After the 45-minute incubation, place the plates in a Beckman tabletop centrifuge and spin at
3150 rpm for 1 hour at 10C.
Promptly remove plates after the spin, remove rubber cover and place folded paper towel
over the tops of the plate. Quickly, invert the plate onto the paper towel, place it back in the
centrifuge and bring it up to 1000 rpm and quickly shut if off again. This will pull the rest of
the iso-propanol out of the plate and will leave the purified PCR product behind. DON’T let
the spin go above 1000rpm, DON’T wait too long after the spin to do the inversion, and
DON’T let it spin at 1000rpm for any length of time.
The samples are now ready to be loaded on a gel. Note: We typically never purify the plates
too far in advance. If you want to store a plate for the next day, simply wrap in aluminum
foil and store at 4C.
K. L. Kennedy
1
Note: We use Genescan and Genotyper for our data analysis. The manuals provide all the
information necessary for doing analysis. Genotyper has the ability to use macros and create
tables in an automated fashion. We have a pipeline in place and would be happy to answer
any questions related to this software.
Contact:
Kathleen L. Kennedy
kkennedy@mcw.edu
414-456-4537
K. L. Kennedy
2