Biomedical Engineering Reference
In-Depth Information
3.2.1. Reagent Preparation
1.
Add absolute ethanol to buffers AW1 and AW2 as directed on the bottles.
2.
Dissolve the protease with the protease solvent and divide the ready protease into
aliquots. Store ready-to-use protease at -20°C until first use. Thaw protease
before use and adjust it to room temperature. After having been thawed once, the
protease should be kept at 4°C, at which it is stable for up to 2 mo.
3.
Heat the water bath to 56°C.
3.2.2. Samples and Labeling
1.
For each sample label one 15-mL tube, one 1.5-mL microfuge tube, and one
QIAamp spin column.
2.
Thaw the protease and adjust it to room temperature.
3.
Thaw samples and adjust them to room temperature.
4.
L. It is best to use the
same volume for all of the samples being analyzed. When using 400
Sample volume (X) can range between 200 and 1000
µ
L or less of
sample, the procedure should be carried out using 1.5-mL microfuge tubes, and
brief spins should be performed before
steps 5
and
6
.
µ
5.
The procedure is carried out at room temperature.
3.2.3. Protocol
1.
Pipet 0.1X
L of protease to each of the 15-mL tubes (or 1.5-mL tubes, if sample
volume in less than 400
µ
µ
L).
2.
Add X
µ
L of plasma to the matching 15-mL tube.
3.
Add X
µ
L of buffer AL and vortex for 15 s.
4.
Incubate the samples in a water bath of 56°C for 10 min.
5.
Add X
µ
L of absolute ethanol to each tube, vortex for 15 s.
6.
L of the mixture on the spin column and centrifuge for 1 min at
8000
g
at room temperature.
Load 630
µ
7.
Transfer the column to a new collecting tube (supplied with the kit). If more than
200
L of sample is processed, this step should be repeated until all of the mix-
ture is loaded on the column. Discard the flow-through before each spin.
µ
8.
L of Buffer AW1 to each column and centrifuge at 8000
g
for 1 min at
room temperature.
Add 500
µ
9.
Transfer the columns into new collecting tubes.
10.
Add 500
µ
L of Buffer AW2 to each column and centrifuge at maximum speed
for 3 min.
11.
Carefully transfer the columns to a clean, labeled, 1.5-mL Eppendorf tube, ensure
that there are no traces of the ethanol in the column, and discard the flow-through
to a special container for sodium azide waste.
12.
Add 50
µ
L of Buffer AE to each column. Incubate for 5 min at room temperature.
13.
Centrifuge at 8000
g
for 1 min at room temperature to elute the DNA from the
columns.
14.
Discard the columns and store the samples at -20°C.
