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Automated PerkinElmer Chemagic 360 Protocol
For isolating DNA from large volume whole blood
Equipment:
• PerkinElmer Chemagic 360, Magnetic Particle Separator Machine
• Eight 12-tube Metal Racks
• One 12-Head Metal Rod Cover Rack
• 65°C water bath
C1
B1
A1
Supplies and Reagents:
• 50mL tubes: at maximum capacity, 7 metal racks x 12
tubes = a total of 84, 50ml tubes and 1 metal rack holding
12 plastic rod-head covers.
C2
B2
A2
• Chemagic DNA Blood Kit CMG-704
o Lysis Buffer #1 (CMG-704, 2.1Liter, each batch
has a unique lot number)
C3
B3
A3
o Binding Buffer #2 (CMG-704, 2.1 Liter)
o Wash Buffer #3 (CMG-704, 2.1 Liter)
o Wash Buffer #4 (CMG-704, 2.1 Liter)
C4
B4
A4
o Wash Buffer #5 (CMG-704, 2.1 Liter)
o Wash Buffer #6 (CMG-704, 2.4 Liter)
o Protease (CMG-704, reconstituted in 7ml)
Fig1. Depiction of 50mL metal tube rack.
o Magnetic Beads (CMG-704, 320ml)
o Elution Buffer #7 (CMG-704, 240ml, 10mM TrisHCL, pH = 8.0)
Purpose:
This protocol is for isolating genomic DNA from large volume fresh or frozen blood
samples.
Important considerations:
• This protocol can be used for isolating DNA from large blood volumes ranging
from ≥5-11mls.
• Do not touch any part of the machine during operation (once the machine has
started.
• Due to potential contagious pathogens in human blood, wear all personal
protective equipment.
Procedure:
1. Turn on machine and log-in.
2. Determine the number of samples that will be run on the PerkinElmer Chemagic
360. Increments of 4, 8, or 12 samples can be run on the machine at a time.
Procedure continued on next page
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Important:
• Before starting a DNA extraction run, the dispenser manifolds need to be
primed. In the program main window, find the Select Protocol options pulldown menu and select a protocol in Available Protocols named:
“Primed manifolds H12 all”
3. Based on the number of samples planned on being processed, calculate number
of 50ml tubes used in the experiment, in increments of 4 (4, 8, or 12). Uncap the
number of 50mL tubes and place them in the racks. A1 is the starting position.
Place this tube in the rack first and add the other tubes in sequential order: First
A1-A4 = 4 samples, next B1-B4 = 8 samples, next C1-C4 = 12 samples
(Please see Fig1).
4. Put all patient identification cards (PIC) and matching blood samples in the same
numerical order.
Important:
• Open DNA Lab program in the main lab computer and update the status
of the samples.
5. ITBioPath samples do not need to be
updated.Print two bar-coded labels from
the Zebra printer. Place these bar-code
Rack 5
stickers on two 50mL tubes for each
Rack 4
sample (one for Input 50ml tube and one
Rack 6
for Output 50ml tube).
6. On the PerkinElmer machine turntable,
place three metal racks (containing empty
Rack 3
50ml tubes), into Rack Slots 4, 5, and 6
Rack 7
(Please see Fig2).
7. The 12-Head Metal Rod Cover Rack is
Rack 2
located in Rack Slot 1. Similar to the 50ml
Rack 8
tubes, place the appropriate number of
plastic rod head covers into Rack 1 and
Rack 1
place the Rack onto Slot 1 on the
turntable.
8. Pipette 900µl of magnetic beads into each
Fig2. Depiction of Chemagic 360 turntable from above.
50ml tube of a single rack. Place that rack
in Rack Slot 3.
9. Pipette 50µl of protease into the bottom of 50ml Input Tubes (Rack 2).
10. In the same Rack 2 (containing 50ml protease), transfer blood in vacutainer
collection tubes by carefully pouring them into the 50mL Input Tubes. Place that
rack in Rack Slot 2.
Procedure continued on next page
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11. Pipette 1ml of elution buffer into the 50ml Output Tubes. Place that rack in Rack
Slot 7.
Important!
• Make sure that the 50ml Input and Output tubes are in the same
corresponding position before you start the protocol.
12. Important!
• In the program main window, there is a Select Protocol options pull-down
menu. You must select the protocol file named:
“Chemagic DNA Blood10K 360 drying prefilled VD150513.che”
Note:
• This protocol was specifically designed for processing large blood
volumes of up to 11mls of whole blood. Other protocols have different
purposes and have been developed for other sample types and
conditions. A second CTRC team member must double-check the correct
protocol has been selected.
13. Insert sample ID’s into the PerkinElmer computer by scanning the 50ml Output
Tubes. The Input and Output tubes have the same bar-code name and the barcodes/tubes need to be scanned once.
14. When the pop-up box appears for selecting the buffer identification, select “No”
to skip this step.
15. Close the Chemagic 360 safety cover on the PerkinElmer machine. Press
continue and start.
16. Fill out the PerkinElmer Run check list taped to the side of the machine.
17. When the extraction run has finished, examine all racks and tubes for unusual
problems. If something appears abnormal, ask Dan Winder to inspect the
machine. If everything looks normal, recap the 50ml Output Tubes in Rack Slot
7.
18. Incubate the 50ml Output Tubes (containing DNA) in a 65°C water bath for 2060 minutes.
19. After the incubation period, place the 50ml Output Tubes, with caps, in the
laminar biosafety cabinet rotating platform for 10 minutes to allow excess ethanol
to evaporate.
20. To avoid leakage, ensure the tube caps are tightly closed. Place the 50ml
Output Tubes on rotating platform until samples are ready for quality control.
Note: The DNA can now be quantified using the Nanodrop
(spectrophotometer). After Nanodrop measurements are taken, samples are
centrifuged briefly and transferred to a storage tube.
Protocol:
Developed by PerkinElmer and modified by the Cellular Translational Research
Core, Dept. Center for Clinical & Translational Science, University of Utah.
Last updated: 9-1-2016