Download VIII. Purification of labeled probes using the High Pure PCR Product

Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes
VIII. Purification of labeled probes
using the High Pure PCR Product
Purification Kit
The High Pure PCR Product Purification
Kit is designed for the efficient and convenient isolation of PCR products from amplification reactions, but is also suited for the
removal of unincorporated nucleotides from
DIG DNA and RNA labeling reactions.
The DNA binds specifically to the surface of
glass fibres in the presence of chaotrope
salts. Primers, unincorporated nucleotides,
contaminating agarose particles and proteins
are removed by a simple washing step. The
bound DIG-labeled DNA is subsequently
eluted in a low-salt buffer.
Note: A minimum length of approx. 100 bp
is required for efficient binding. The kit
can therefore not be used for the removal
of unincorporated nucleotides from oligonucleotide labeling reactions.
4
2 Add 500 µl binding buffer (vial 1, green
Q
cap) and mix well.
Note: It is important that the volume
ratio between sample and binding buffer
is 1: 5. When using other sample volumes
than 100 µl, adjust the volume of binding buffer accordingly.
Q
3 Insert a High Pure filter in a collection
tube and pipette the sample into the upper
buffer reservoir.
4 Centrifuge at 13.000 x g in a microQ
centrifuge for 30 sec.
5 Discard the flow through and combine
Q
the filter tube again with the same collection tube.
6 Add 500 µl wash buffer (vial 2, blue cap)
Q
to the upper reservoir and centrifuge as
in step 4.
Q
7 Discard the wash buffer flow through
and recombine the filter tube again with
the same collection tube.
8 Add 200 µl wash buffer (vial 2, blue cap)
Q
to the upper reservoir and centrifuge as
in step 4.
Products required
*This product is sold under licensing
arrangements with Roche
Molecular Systems and The PerkinElmer Corporation. Purchase of this
product is accompanied by a
license to use it in the Polymerase
Chain Reaction (PCR) process in
conjunction with an Authorized
Thermal Cycler. For complete
license disclaimer, see inside back
cover page.
Reagent
Description
Available as
High Pure PCR Product
Purification Kit*
Kit for 50 purifications
Kit for 250 purifications
Cat. No. 1 732 668
Cat. No. 1 732 676
consisting of:
• Binding buffer,
green cap
• Wash buffer,
blue cap
• Elution buffer
• High Pure filter tubes
• Collection tubes
nucleic acids binding buffer; 3 M guanidine-thiocyanate, • Vial 1
10 mM Tris-HCl, 5% (v/v) ethanol, pH 6.6 (25° C)
wash buffer; add 4 volumes of absolute ethanol
• Vial 2
before use! Final concentrations; 20 mM NaCl,
2 mM Tris-HCl, pH 7.5 (25° C), 80% ethanol
elution buffer; 10 mM Tris-HCl, 1 mM EDTA,
• Vial 3
pH 8.5 (25° C)
Polypropylene tubes, containing two layers of a
specially pre-treated glass fibre fleece; maximum
sample volume: 700 µl
2 ml polypropylene tubes
Procedure
Note: Make sure that 4 volumes ethanol
have been added to the wash buffer (vial 2,
blue cap). The binding buffer (vial 1,
green cap) contains guanidine-thiocyanate
which is an irritant. Wear gloves and
follow laboratory safety conditions during
handling.
Q
1 Fill up the labeling reaction to 100 µl
with redistilled water.
50
Q
9 Discard the collection tube and insert
the filter tube in a clean 1.5 ml reaction
tube (not provided).
10 Add 50–100 µl elution buffer (vial 3) or
Q
redist. water (pH 8.0–8.5) to the upper
reservoir for the elution of the DNA.
Centrifuge as in step 4.
Note: The elution efficiency is increased
with higher volume of elution buffer
applied. At least 68% and 79% recovery
are found with 50 and 100 µl elution
buffer, respectively. Normally, almost
quantitative recovery can be found, as can
be determined in a direct detection assay.
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