CSI-TRU
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Updated: 2/18/2016
DNA Extraction
Promega DNA-IQ System
Database Protocol
Rooted hair samples
Always wear gloves and use barrier (filter) tips when performing this protocol. To
prevent confusion and contamination, never process the hair from more than 4 donors at
one time.
Preparation:
Before beginning, put down a fresh piece of bench paper over the area in which you will be
working and then make sure you have all of the following on the work table:
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Your personal DNA extraction kit
Clean microscope slides and cover slips
Several sterile scalpels
Ice bucket with ice (or freezer tube holder)
Box of gloves (your size)
A box of filter tips (small size)
Plastic tube rack
1 tube of 10 mg/mL proteinase K from your personal freezer box (placed in tube rack
to thaw)
1 tube 1 M DTT from your freezer box (placed in tube rack to thaw)
1 tube Incubation Buffer from your freezer box (placed in tube rack to thaw)
No more than 4 CSI-TRU hair tubes (taken from the freezer)
Pair of tweezers
Container of sterile 1.5 mL microcentrifuge tubes
DNA-IQ magnetic stand
10% bleach solution
Biohazard waste container
Set of micropipettors
Calculator
Timer
Ultra-fine point sharpie marker
Lab notebook
Gel pen for writing in lab notebook
Also check to make sure that following equipment is available and in working order:
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Water bath set at 56 degrees C
Tube heater set at 65 degrees C with 1.5-mL tube block in place
Microcentrifuge
Vortexer
Microscope
Procedure:
1.
Turn on the water bath and tube heater (if not already on).
CSI-TRU
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Updated: 2/18/2016
2.
Remove your donor tubes containing hair from the freezer and place them on the
bench near the microscope.
3.
Open the first donor tube and examine each hair under the microscope, placing
the root-end of the hair on a clean microscope slide and covering it with a cover slip
to prevent movement during examination. If you can't tell which end has the root,
examine both ends under the microscope until you can determine the hair's
orientation.
4.
Cut the end of each hair about 1 cm from the root using a sterile scalpel. Then use
your tweezers to transfer the root ends into a clean, labeled 1.5-mL microcentrifuge
tube.
Although you will examine and cut all the hairs in each donor's tube, retain
half the roots in case you need them later. These ends should be
placed in a sterile 1.5-mL microcentrifuge tube that is clearly labeled with
the TRU-Sample # and returned to the collection bag for that donor (in the
freezer).
5.
Calculate the total amount of incubation/DTT/proteinase K buffer you will need
according to the following formula (per sample):
Incubation buffer
1 M DTT
proteinase K solution
80 uL
10 uL
10 uL
100 uL
For example, if you are planning to process the hair from four donors, you would
need to make up 400 uL of this buffer.
6.
Make up the incubation/DTT/proteinase K buffer and gently mix it by inverting
several times. Then store it on ice or in a freezer tube holder throughout
the remainder of the procedure.
7.
Add 100 uL incubation/DTT/proteinase K buffer to each of the 1.5-mL tubes
containing the donor hair roots. Invert several times to mix and then incubate the
tubes in the 56 degree water bath for 1 hour. Make sure that the hairs are
completely covered by the buffer during this incubation. You can use a
sterile toothpick to push the hairs under the buffer solution, if needed.
8.
While you are waiting, calculate the total amount of lysis buffer you will need to
prepare by multiplying the number of hair donors by 300 uL. (For 4 hair donor tubes,
1,200 uL of lysis buffer would be needed.)
9.
Invert the tube of lysis buffer (DNA extraction kit) several times to mix and then
aliquot the calculated volume into a sterile 1.5-mL microcentrifuge tube.
10.
Mix the 1 M DTT by vortexing for a few seconds. Then add 1 uL of 1 M DTT for every
100 uL of lysis buffer.
11.
Mix the lysis buffer and DTT together by inverting the tube several times.
CSI-TRU
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Updated: 2/18/2016
12.
When the incubation of the tubes is complete, remove them from the water bath
and add 200 uL of lysis buffer/DTT mixture and 7 uL of DNA-IQ resin to each tube.
(Make sure to vortex the resin to ensure that it is well-mixed before aliquoting.)
13.
Vortex the tubes at high speed for 3 seconds and then incubate at room temperature
for 5 minutes.
14.
Vortex the tubes at high speed. The place then in the magnetic stand. Separation of
the resin from the surrounding solution should occur instantly. If the resin does
not form a distinct pellet on the side of the tube, vortex the tube and quickly
place it back in the stand.
15.
Using a micropipettor, carefully remove and discard the solution from each tube,
being careful not to disturb the resin pellet (which is now bound to the DNA).
16.
Add 100 uL of lysis/DTT buffer to each tube. Remove the tubes from the magnetic
stand and vortex for 2 seconds at high speed.
17.
Return the tubes to the magnetic stand. Carefully remove and discard all of the lysis
buffer, being careful not to disturb the resin pellet.
18.
Add 100 uL Wash Buffer to each tube. Remove the tubes from the magnetic stand
and vortex for 2 seconds at high speed.
19.
Return the tubes to the magnetic stand and remove and discard all of the Wash
Buffer.
20.
Repeat steps 18-19 two more times for a total of 3 washes. Make sure that all of the
solution has been removed from each tube after the last wash.
21.
With the tubes in the magnetic stand the lids open, air-dry the resin for 5 minutes.
Do not dry the resin for more than 20 minutes, as this may inhibit the
removal of DNA.
22.
Add 50 uL of elution buffer (DNA extraction kit) to each tube.
23.
Close the lids and vortex for 2 seconds at high speed. Then incubate the tubes in the
65 degree tube heater for 5 minutes.
24.
Remove the tubes from the heater and vortex for 2 seconds at high speed. Then
immediately place the tubes in the magnetic stand. The tubes must remain hot as
they are placed in the magnetic stand, or yield will decrease.
25.
Using a micropipettor, carefully transfer the DNA-containing solution to a sterile 1.5mL centrifuge tube. Make sure the tubes are labeled with the appropriate TRUSample #, followed by the letter "H" (for hair). For example, if you are processing
TRU-89, you should label the top and side of the tube "TRU-89-H".
26.
Place the tubes (now containing the extracted DNA) in your personal freezer box and
place the box back in the freezer.
CSI-TRU
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Updated: 2/18/2016
Clean-up:
1.
After you are done, be sure to put your DNA extraction kit away.
2.
Then discard all debris (used tubes, gloves, used toothpicks, etc.) in the trash.
3.
If you used up any items (e.g. beaker of sterile 1.5-mL tubes, box of filter tips),
replace them from the supply shelf for the next person.
4.
Be sure to turn off the water bath and lights and close the door firmly behind you
when you leave.