| Description |
| The RT² SYBR Green / Fluorescein qPCR master mix contains all
of the reagents and buffers required for real-time polymerase chain
reactions in the BioRad iCycler® and MyiQ®: real-time PCR buffer, a
high-performance HotStart DNA Taq polymerase, nucleotides, SYBR® Green
dye, and the Fluorescein reference dye needed to normalize the
instruments' optics. Simply add the master mix to PCR tubes along with
your template and primers. The chemically-modified and tightly controlled
HotStart enzyme uniquely provides more accurate SYBR Green results by
preventing the amplification of primer dimers and other non-specific
products. This RT² qPCR master mix is best suited for
real-time PCR applications using SYBR Green based detection on the BioRad
iCycler® and MyiQ®. |
| Materials Included /
Packing List |
|
Please check the kit components
immediately after you receive this package. SABiosciences is not responsible
for any missing items not reported within two (2) business days upon
receipt.
Contents:
PA-011: Two (2) tubes each containing 1.4 ml of 2X solution and enough for 100 standard 25-µl reactions
PA-011-12: Twelve (12) tubes each containing 1.4 ml of 2X solution and enough for one PCR Array
PA-011-24: Twenty-four (24) tubes each containing 1.4 ml of 2X solution and enough for one PCR Array
PA-111: One (1) bottle containing 25 ml of 2X solution and enough for 2000 standard 25-µl reactions
Storage Conditions: The kit is shipped on dry ice or cold packs.
For long-term storage, keep at -20 ºC. If entire volume is not be used all at once, divide into
aliquots and store at -20 ºC.
Avoid repeated freezing and thawing.
Shelf Life: All reagents are stable for 6 months after receipt if
stored at the recommended temperature. |
| Brief Protocol |
- Ensure that you do not contaminate the 2X real-time PCR cocktail by
dividing into aliquots containing the amount of cocktail necessary for
the number of reactions you are preparing each day. The rest of the
cocktail should be kept in storage away from any sources of template
DNA.
- When using RT² qPCR Master Mixes with RT² Profiler™ PCR Arrays or RT²
qPCR Primer Assays or ChIP-qPCR Primer Assays, refer to the appropriate User Manuals.
- For general real-time PCR purposes, mix the following components in a PCR
tube:
- 12.5 µl RT² SYBR Green / Fluorescein qPCR Master Mix
- 10.5 µl ddH2O
- 1.0 µl template cDNA (up to 250 ng)
- 1.0 µl gene-specific 10 µM PCR primer pair stock
25.0 µl final volume
- Recommended real-time thermal cycler programs:
95 ºC, 10 min; 40 cycles of
(95 ºC, 15 sec; 60 ºC, 60 sec).
NOTE: The 10 min step at 95 ºC is required to activate
the HotStart Taq DNA polymerase.
- Program the real-time thermal cycler to detect and record the
SYBR® Green signal from every reaction at the end of the 60 ºC
annealing / extension step of each cycle.
- Run your instrument's default melting curve program immediately
after the above PCR program, and generate a first derivative
dissociation curve. No more than one peak should appear in each
reaction
If your instrument does not have a default melting curve program, run
the following program instead:
95 ºC, 1 min; 65 ºC, 2 min (OPTICS OFF); 65 ºC to 95 ºC
at 2 ºC / min (OPTICS ON).
NOTE: Be sure to visually inspect the plate after the run for any
signs of evaporation from any of the wells. If evaporation is observed, make a
note of which wells so that you may qualify your data analysis appropriately.
SYBR® is a registered trademark of Molecular Probes, Inc.
iCycler® and MyiQ® are registered trademarks of Bio-Rad Laboratories, 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.
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