2012 Promega catalogue

Life
Science
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2012
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Cell Signaling
214
ImProm-II Reverse Transcription System
Product Size Cat.# Price ($)
ImProm-II Reverse Transcription System 100 reactions A3800 668.00
Available Separately
ImProm-II Reverse Transcriptase 10 reactions A3801 64.00
100 reactions A3802 324.00
For Laboratory Use.
500 reactions A3803 1208.00
Description: The ImProm-II Reverse Transcription System produces efficient,
robust synthesis of first-strand cDNA in preparation for PCR amplification. The
components of the ImProm-II Reverse Transcription System can be used to
reverse transcribe RNA templates starting with total RNA, poly(A)+ mRNA or
synthetic transcript RNA. The optimized reaction buffer and powerful ImProm-II
Reverse Transcriptase provided in the ImProm-II System together enable
robust, full-length cDNA synthesis for the reproducible analysis of rare or long
messages. The cDNA synthesis conditions have been formulated for standalone
applications or for easy transition to gene-specific target amplification. From
1–20μl of the reverse transcription reaction can be directly amplified using
Taq DNA polymerase in coupled or uncoupled PCR.
Features:
• Full-Length RT-PCR: Reverse transcribe long RNA template up to 8.9kb.
• Microarray-Compatible: May be used for incorporation of regular, Cy ® 3modified,
Cy ® 5-modified, and amino-allyl-modified nucleotides.
• Easy to Use: System provides all reagents for efficient reverse transcription.
• Scalable and Flexible: Use 1–20μl from the initial RT reaction in
subsequent PCR; the optimized buffer allows for coupled RT-PCR.
• RT Provided with 5X Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25°C),
375mM KCl, 50mM DTT. A 25mM MgCl 2 Solution also is included.
• Versatile: Use with your thermostable DNA polymerase of choice.
Storage Conditions: Store at –20°C. Store Positive Control RNA at –70°C.
Protocol Part#
ImProm-II Reverse Transcription System Technical Manual TM236
M 1µg
37°C
First-strand cDNA completed by ImProm-II RT
TTTTTTTT
100ng
–RT
1µg
8.9kb APC gene mRNA
42°C 45°C 50°C 55°C
100ng
–RT
1µg
100ng
–RT
1µg
100ng
–RT
1µg
100ng
–RT
– 940bp
Full-length cDNA synthesis of 8.9kb template over a range of
temperatures using the ImProm-II Reverse Transcription System
as demonstrated by selective amplification of terminal 3´ sequences
in two-step RT-PCR. Entire 8.9kb message must be reverse transcribed by
the ImProm-II RT from the oligo(dT) primer to amplify the terminal 940bp
sequence. Message was amplified from either 1μg or 100ng of total RNA.
Control reactions without the reverse transcriptase are shown (–RT) as well.
Details of experiment available in the ImProm-II Reverse Transcription
System Technical Manual, #TM236.
3291TB07_2A
A. Cy
ImProm-II RT
® 3 Incorporation
B. Cy
ImProm-II RT
® 5 Incorporation
SuperScript ® II RT
SuperScript ® II RT
Incorporation studies of fluorescently labeled nucleotides. ImProm-II
Reverse Transcription System allows for high-efficiency incorporation
of Cy ® 3 and Cy ® 5 fluorescent nucleotides. This demonstrates fluorescent
nucleotide incorporation by ImProm-II RT vs. SuperScript ® II RT using a
1.2kb kanamycin transcript as template. A single fluorescent band is
produced and visualized using an FMBIO ® II Fluorescence Imaging System.
Relative Fluorescence Units
(in thousands)
A. Cy
100
20
® 3 Incorporation B. Cy ® 5 Incorporation
50
0
ImProm-II
System
SuperScript ® 0
-II ImProm-II SuperScript
System System
3395MB04_2A
® -II
System
Relative Cy ® 3 and Cy ® 5 nucleotide incorporation by ImProm-II
Reverse Transcription System in comparison to Superscript ® II First
Strand Synthesis System. Results with Cy ® 3 dUTP (Panel A) and Cy ® 5
dUTP (Panel B) incorporation are reported. Panels A and B correspond to
Panels A and B in the figure above.
A. B.
A comparison of typical 21-by-21 feature array blocks hybridized with
ImProm-II- or SuperScript ® -II-generated fluorescent cDNA probes.
Panel A. Array hybridized with ImProm-II-generated fluorescent cDNA
probes. Panel B. Array hybridized with SuperScript ® -II-generated fluorescent
cDNA probes. Image brightness was normalized to yield similar background
intensities. See Kasler et al. Promega Notes 81, 14–15, for experimental
details.
For complete and up-to-date product information visit: www.promega.com/catalog
10
3392TC05_1A
3698TA04_2A