Biotin RNA Labeling Mix, 10 × conc. ␮l 40

For general laboratory use.
FOR IN VITRO USE ONLY.
Biotin RNA Labeling Mix, 10 × conc.
RNA labeling with biotin-16-UTP by in vitro transcription with SP6, T7 and T3 RNA polymerases
for 20 transcriptions
Cat. No. 11 685 597 910
40 ␮l
Version Nov. 2004
Store at ⫺15 to ⫺25° C
1. Product overview
Contents
Sufficient for 20 transcriptions.
Label
Biotin RNA
Labeling Mix,
10 × conc.
2. Procedures and required materials
Content
• 40 ␮l
• 10 × conc. NTP labeling mixture: 10 mM ATP, 10 mM
CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM Biotin-16-UTP, pH
7.5 (20°C)
Labeling principle Biotin-16-UTP is incorporated by SP6, T7 and T3 RNA
polymerases at approx. every 20-25th nucleotide of the
transcript under the conditions described below (1).
The Biotin RNA Labeling Mix is especially designed for
the use with Roche Biochemicals SP6, T7 and T3 RNA
polymerases, which are supplied with an optimized
transcription buffer.
Sample material
Application
• linearized plasmid DNA
The DNA to be transcribed is cloned into the polylinker
site of an appropriate transcription vector which
contains adjacent to the polylinker a promoter for
SP6, T7 or T3 RNA polymerase (2,3).
For the synthesis of 'run off' transcripts the plasmid is
linearized by a restriction enzyme. Restriction enzymes
creating 5'-overhangs should be used; 3’ overhangs
should be avoided. The linearized template DNA
should be purified by phenolchloroform extraction and
ethanol precipitation, to avoid RNase contamination.
For 'run around' transcription circular plasmid DNA is
used.
• PCR product
PCR-fragments which contain RNA polymerase
promoter sequences can also act as templates for
transcription. Purification of the correct PCR fragment
by gel electrophoresis prior to transcription is
recommended.
Biotin-labeled RNA is used for hybridization to
• Northern blots
• Southern blots
• plaque or colony lifts or
• RNase protection experiments
• in situ hybridization
Labeling
efficiency
In the standard reaction about 10 ␮g ‘full length’ biotin
labeled RNA is synthesized from 1 ␮g linearized
plasmid DNA with an insert of approx. 1 kb. Larger
amounts of biotin-labeled RNA can be obtained by
scaling up the reaction components.
The amount of synthesized labeled RNA depends on
the amount, size (site of linearization) and purity of the
template DNA.
Note: Longer incubations do not increase the yield of
labeled RNA.
Stability
The unopened vial is stable at ⫺15 to ⫺25° C through
the expiration date printed on the label.
Note: Repeated freezing and thawing should be
avoided. To avoid contamination we recommend to
aliquot the Biotin RNA labeling Mix solution and to
store in 2-3 portions.
1104.11693 069 ➅
2.1 Standard labeling assay
Additional
• SP6 RNA polymerase* or
equipment and
• T7 RNA polymerase* or
reagents required
• T3 RNA polymerase*
• DNase, RNase free* (optional)
• Transcription buffer 10 × conc., is supplied with the
RNA polymerases: 400 mM Tris-HCl, pH 8.0 (20° C);
60 mM MgCl, 100 mM dithiotreitol (DTT),
20 mM spermidin.
Procedure
Standard labeling assay.
Note: Please make sure, you work under RNase-free
conditions.
Step
1
Action
• Add the following to a microfuge tube on ice:
Reagent
Volume
1 ␮g linearized plasmid DNA or appropriate
amount of PCR product (100-200 ng)
x ␮l
Biotin RNA labeling mix, 10 ×
2 ␮l
10 × transcription buffer
2 ␮l
add sterile RNase free double dist. water to
a final volume of 18 ␮l
x ␮l
RNA polymerase (SP6, T7 or T3)
2 ␮l
• Mix and centrifuge briefly.
• Incubate for 2 h at 37°C.
2
• Add 2 ␮l DNase I, RNase-free to remove template DNA.
(optional) • Incubate for 15 min at 37° C.
Note: Only required for RNase-protection experiments!
3
Stop the reaction by adding 2 ␮l 0.2 M EDTA (pH 8.0).
4
Use the labeled probe immediately or store ethanol precipitated at
⫺15 to ⫺25° C or ⫺60° C or below.
DNase treatment
When the biotin-labeled RNA is used for hybridization
to Northern or Southern blots, plaque or colony lifts a
DNase-treatment is not required, as the amount of
biotin-labeled RNA transcript is far in excess of the
template DNA.
2.2 General remarks on usage and application
Analysis of
labeled RNA
Quality and quantity of the transcript can be analyzed
by non-denaturing agarose gel electrophoresis and
ethidium bromide staining. The signal from the RNA
band should be stronger than that from the DNA. The
size and the amount of the transcript can be estimated
by comparison to known RNAs.
Hybridization with Add 0.2-1 ␮l (approx. 20 -100 ng) of the biotin-labeled
labeled RNA
RNA per ml hybridization solution.
Detection of
biotin-labeled
RNA probes
After hybridization to nucleic acid targets bound to
nylon membrane, the biotin label is detected by an
immunoassay with anti-biotin-AP in the Biotin
Luminescent Detection Kit*, or Streptavidin-AP
conjugate and the color substrates NBT/BCIP* or the
chemiluminescent substrates CSPD1) or CDP-Star2).
For in situ hybridizations, avidin, conjugated to
fluorophores is used.
Single reagents
Product
Pack Size
Anti-biotin-AP, Fab fragments
11 426 303 001
Avidin-Fluorescein
1 mg
11 975 595 910
Avidin-Rhodamine
1 mg
11 975 609 910
CDP-Star, ready-to-use
2x 50 ml
12 041 677 001
CSPD, ready-to use
2x 50 ml
11 755 633 001
DIG RNA Labeling Mix
40 ␮l
11 277 073 910
DIG-labeled control RNA
50 ␮l
11 585 746 910
10 000 units
10 776 785 001
Hybridization bags
50 bags
11 666 649 001
Klenow Enzyme
100 units
500 units
11 008 404 001
11 008 412 001
DNase I, RNase free
NBT/BCIP Stock Solution
Nylon Membrane, positively
charged
3. Appendix
3.1 References
1 Höltke, H.J. & Kessler, C. (1990) Nucleic Acid Res. 18, 5843.
2 Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477.
3 Kasavetis, G. A. et al. (1982) J. Biol. Chem. 257, 5779
3.2 Ordering Information
Protector RNase Inhibitor
DIG Wash and
Block Buffer Set
Biotin Luminescent Detection
Kit
Pack Size
Cat. No
1 kit
11 175 025 910
(2 × 10 labeling
reactions)
30 blots
11 585 762 001
(10 × 10 cm2)
1 kit (50 blots) 11 811 592 910
8 ml
11 681 451 001
10 sheets
(20 × 30 cm)
20 sheets
(10 × 15 cm)
1 roll
(0.3 × 3 m roll)
11 209 272 001
10 000 units
2 000 units
03 335 402 001
03 335 399 001
11 209 299 001
11 417 240 001
SP6 RNA polymerase
1000 units
10 810 274 001
T3 RNA polymerase
1000 units
5000 units
11 031 163 001
11 031 171 001
T7 RNA polymerase
1000 units
5000 units
10 881 767 001
10 881 775 001
Kits
Product
DIG RNA Labeling Kit (SP6/T7)
Cat. No.
150 units
(200 ␮l)
*available from Roche Applied Science.
1)
CSPD is a trademark of Tropix, Inc. Bedford, MA, USA and covered by European patent application 0497 972 and U.S. patent 5112960, both assigned to Tropix Inc.
2)
CDP-Star is a trademark of Tropix, Inc., Bedford, MA, USA and covered under U.S. patent
5,326,882.
DIG-11-UTP is covered by the following patents,
EP patent 0324474 and US 5,344,757 granted to Roche
Applied Science
How to contact Roche Applied Science
www.roche-applied-science.com
to order, solve technical queries, find product information,
or contact your local sales representative.
www.roche-applied-science.com/pack-insert/11685597910a.pdf
Please visit our new Online Technical Support Site under
www.roche-applied-science.com/support
Roche Diagnostics GmbH
Roche Applied Science
Nonnenwald 2
82372 Penzberg
Germany