R-Biopharm RIDAGENE CMV PG9005 Bedienungsanleitung Seite 30

8.2 DNA isolation from urine
For DNA isolation from urine, specimens must be used that were collected in
preservative-free containers according to laboratory guidelines and were not stored
and/or transported for longer than 4 hours at room temperature (+18 °C to +25 °C).
Alternatively, the specimens can be stored for up to 3 days at +2 °C to +8 °C.
Aliquots of the specimens can be stored for up to 30 days at -20 °C. Specimens
stored for longer than 30 days must be kept at -70 °C. It is recommended to produce
aliquots of the specimens to avoid repeated thawing and freezing.
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The RIDA GENE CMV test contains an Internal Control DNA that can be used as
an extraction control for specimen preparation and inhibition control. To use the
Internal Control DNA , you must use 10 µl of the Internal Control DNA during the
extraction.
For purification using the Maxwell® RSC (Promega), use 300 µl urine and mix with
300 µl lysis buffer and 30 µl proteinase K. Incubate in the thermoshaker at 45 °C for
10 min. Transfer everything into the Maxwell cartridge. Add 10 µl Internal Control
DNA . Pour 60 µl nuclease-free water into the vials provided. On the device, select
the "Maxwell® RSC Viral Total Nucleic Acid" protocol.
For purification with MagNA Pure 96 (Roche), use 200 µl urine and pipette 10 µl of
Internal Control DNA into the MagNA Pure 96 Processing Cartridge. Prepare using
the Pathogen Universal 200 protocol. Elute in 50 µl.
9. Test procedure
9.1 Preparation of the PCR mix for qualitative detection
The total number of the reactions needed for PCR (samples and control reactions)
must be calculated. One positive and one negative control must be included in each
test run.
Pipette 20 µl of the master mix into each reaction vial (vial/plate).
Negative control:
Sample:
Positive control:
Seal the reaction vials or plates, briefly centrifuge at slow speed, and transfer into the
real-time PCR device. Start PCR according to device settings (see Table 3).
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Add 20 µl PCR water to the pre-pipetted master mix as a
negative control.
Add 20 µl DNA extract to the pre-pipetted master mix of
the sample reactions.
Add 20 µl Positive Control to the pre-pipetted master
mix in the designated reaction vial.
2019-05-28
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RIDA GENE CMV