CSI-TRU
QIAGEN DNA Micro Kit
Blood Extraction Protocol
Things You Will Need:
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Heating block set at 56 degrees C with 1.5-mL tube block in place
Heating block set at 70 degrees C with 1.5-mL tube block in place
Your CSI-TRU donor blood cards
Sterile scalpels (one per DNA extraction)
Beaker of 1.5 mL microcentrifuge tubes (sterile)
Microfine Sharpie to label your tubes
Chemical waste beaker labeled "Chaotropic Salts from QIAGEN kits"
QIAGEN DNA Micro Kit containing:
Buffer AL
Buffer AW1
Buffer AW2
Buffer AE
Carrier RNA (kept on ice)
QIAamp MiniElute Columns
Buffer ATL
Proteinase K
Prepare Blood Cards:
1.
For optimal DNA extraction, use a fresh scalpel and cut three 3-mm2 blood
samples from your card (a total of 9-mm2). However, don't take more than half of
what you have left (in case you mess up during the extraction - always remember
the Rule of Solomon!) If you don' t have enough blood on your card to take 9 mm2
and still have half left, take half of whatever you have and carefully record the
total area you removed. (This will affect the volume of buffer you use at the end
of the extraction to elute your DNA from the column.)
2.
Cut your blood sample(s) into smaller pieces to maximize contact with the
buffers. Then place the cut pieces into a 1.5 mL tube labeled with your CSI-TRU
donor sample number.
3.
Prepare ATL/Proteinase K buffer:
180 µL Buffer ATL for each sample (e.g. 180 x 4 samples = 720 µL)
20 µL Proteinase K for each sample (e.g. 20 x 4 samples = 80 µL)
Vortex to mix.
4.
Aliquot 200 µL of ATL/Proteinase K buffer into each sample tube. Mix
thoroughly by vortexing at high speed for 3 seconds.
5.
Place sample tubes into the 56oC heating block and incubate for one hour.
Vortex sample tubes on high speed for 10 seconds every 10 minutes.
Votexing will improve lysis.
6.
Between vortexings, prepare the Buffer AL/carrier RNA:
200 µL Buffer AL for each sample (e.g. 200 x 4 samples = 800 µL)
1 µL Carrier RNA for each sample (e.g. 1 x 4 samples = 4 µL)
Votex to mix then keep on ice until ready to use.
7.
After the last 10 second vortex, briefly centrifuge the sample tubes to remove
drops from the lid.
8.
Aliquot 200 µL Buffer AL/carrier RNA into each sample tube. Mix thoroughly
by vortexing on high speed for 3 seconds. A precipitate may form; however, it
will dissolve when heated and will not interfere with the procedure.
9.
Incubate the tubes in the 70o C heating block for 10 minutes. Votex on high
speed for 10 seconds every 3 minutes.
10.
Vortex a final time and then briefly centrifuge the sample tubes to remove drops
from the lids.
11.
Transfer the lysates directly to labeled QIAamp columns, being careful not to wet
the rims. Close the caps and centrifuge at 8,000 rpm for 1 minute. Then place the
QIAamp columns into clean 1.5 mL microcentrifuge tubes. Discard the
microcentrifuge tubes containing the flow through.
12.
Add 500 µL Buffer AW1 to each column, being careful not to wet the rim.
Centrifuge at 8,000 rpm for 1 minute. Transfer the columns to a clean 1.5 mL
microcentrifuge tubes and discard the tubes containing the flow through as
chemical waste.
13.
Add 500 µL Buffer AW2 to each column, being careful not to wet the rim.
Centrifuge at 8,000 rpm for 1 minute. Transfer the column to a clean 1.5 mL
microcentrifuge tube and discard the tubes containing the flow through as
chemical waste.
14.
Place the column into a fresh 1.5-mL microcentrifuge tube and centrifuge at full
speed (14,000 rpm) for 3 minutes to completely dry the column membrane.
Check the column to make sure the column contains no more buffer. If any
visible buffer remains, centrifuge again. The column must be “dried” because the
ethanol in the buffer may interfere with downstream applications if it remains
with the DNA as it is eluted from the columns. Discard the tubes containing the
flow through as chemical waste.
15.
Place the columns into clean, labeled 1.5 mL microcentrifuge tubes. Carefully
add Buffer AE using the volume appropriate for the original size of the blood
sample card taken as follows:
Total Size of Sample
9 mm2
6 mm2
3 mm2
Volume Buffer AE
100 µL
50 µL
30 µL
If your sample was less than 3 mm2, use the minimum volume of 20 µL for your
elution volume. Dispense the Buffer AE onto the center of the columns, being
careful not to touch the membrane or wet the sides.
16.
Incubate the columns at room temperature for 5 minutes. Then centrifuge at
14,000 rpm for 2 minutes. If the Buffer AE did completely flow though the
column, centrifuge again. Dispose of the column as chemical waste and store
the flow-through, which contains your DNA extract, in the freezer until ready to
use.