Please check the kit components immediately after you receive this
package. SABiosciences is not responsible for missing items not reported within
two (2) business days upon receipt.
Storage Conditions: The
kit is shipped on dry ice or cold packs. For long-term storage, keep at -20ºC. If entire volume is not be used all at once, divide into aliquots and
store at -20ºC. Avoid repeated freezing and thawing.
Shelf Life: All reagents are
stable for 6 months after receipt of the kit if stored at the recommended
AmpH-024: One (1) tube containing
90µl RT² PreAMP cDNA Human Angiogenesis Primer Mix solution
Enough for 12 25-µl standard PreAMP reactions
Read carefully the User Manual of the RT² PreAMP cDNA Synthesis Kit
prior to first-time use. Refer to the instructions in the User Manual
every time you use the RT² PreAMP cDNA Synthesis Kit (catalog #330451).
Ensure that you do not contaminate the RT² PreAMP cDNA Synthesis
Primer Mix by using a fresh pipette tip every time you draw an aliquot
from the tube.
- Perform first strand synthesis using the RT² PreAMP cDNA Synthesis Kit as
A. Add 2 Ál of GE to 8 Ál of RNA (1-100ng). Incubate at 42░C for 5 min and
immediately chill on ice.
B. Mix a master mix for the RT reaction as below:
|For 1 reaction
|BC3 ||4 µl
|RE ||1 µl
|RI ||1 µl
|P2 ||1 µl
|RNase/DNase free H2O ||3
C. Add 10 µl of the RT master mix to 10 µl GE-treated RNA.
D. Incubate at 42ºC for 30 min and heat at 95ºC for 5 min. Chill on ice or
store at -20ºC until proceeding to the next step.
For the normal standard reaction, mix the following components in a PCR
|| 2X RT² PreAMP PCR Master Mix
|| RT² PreAMP cDNA Primer Mix specific for the
RT² Profiler™ PCR Array of your choice
|| Template undiluted cDNA from the 20-µl first
strand synthesis reaction (step 2D)
|| final volume
Perform 12 cycles of PCR in a thermal cycler:
NOTE: The 10 min step at 95ºC is required to activate the HotStart Taq
95ºC, 10 min; 12 cycles of (95ºC, 15 sec; and 60ºC, 2 min); 4ºC
Add 2 µl of the Side Reaction Reducer to each pre-amplified reaction, and
incubate at 37ºC for 15 min followed by heat inactivation at 95ºC for 5
Dilute the 27-µl pre-amplified templates to 111 µl by adding 84 µl of dd
H2O. For immediate use, keep on ice prior to loading onto the RT² Profiler™
PCR Array, or store at -20ºC until ready.
For use in the RT² Profiler™ PCR Array, prepare the Experimental Cocktail with the following components in
15-mL conical tube:
||2X SABiosciences RT² qPCR SYBR Green Master Mix (Note:
Use the appropriate master mix specific for your real-time PCR
||diluted PreAMP PCR reaction
|| final volume
Add 25 µl of the above Experimental Cocktail to each well of the PCR Array,
preferably from a reservoir with an eight-channel pipettor (or a
twelve-channel pipettor but only using eight tips).
Run the following real-time thermal cycler program:
NOTE: The 10 min step at 95ºC is required to activate the HotStart Taq DNA
95ºC, 10 min; 40 cycles of (95ºC, 15 sec; and 60ºC, 60 sec)
Program the real-time thermal cycler to detect and record the SYBR« Green I
signal from every reaction during the annealing step of each cycle.
SYBR« is a registered trademark of Molecular Probes, Inc.
Purchase of this product includes an immunity from suit under patents
specified in the product insert to use only the amount purchased for the
purchaser's own internal research. No other patent rights are conveyed
expressly, by implication, or by estoppel. Further information on purchasing
licenses may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.