| Description |
| The RT² SYBR Green / ROX qPCR master mix contains all of the
reagents and buffers required for real-time polymerase chain reactions in
ABI® and Stratagene® instrumentation: real-time PCR buffer, a high-performance HotStart
DNA Taq polymerase, nucleotides, SYBR® Green dye, and the ROX 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 ABI® and Stratagene® instrumentation. |
| 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-012: Two (2) tubes each containing 1.4 ml of 2X solution and enough for 100 standard 25-µl reactions
PA-012-12: Twelve (12) tubes each containing 1.4 ml of 2X solution and enough for one PCR Array
PA-012-24: Twenty-four (24) tubes each containing 1.4 ml of 2X solution and enough for one PCR Array
PA-112: 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 PCR purposes, mix the following components in a PCR
tube:
- 12.5 µl RT² SYBR Green / ROX 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 program:
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.
ROX® is a trademark of Applera Corporation
ABI® s a registered
trademark of Applied Biosystems
Stratagene® is a registered trademark of
Stratagene Corporation.
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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