ReactionReady™ Mouse GAPD Internal Normalizer

Description

The ReactionReady™ Mouse GAPD Internal Normalizer allows researchers without access to real-time equipment to perform semi-quantitative RT-PCR analyses. This unique primer mix is designed to detect the expression level of GAPD, a commonly used housekeeping gene, in the same tube as your gene of interest. Just add the Internal Normalizer to a standard PCR containing your first strand cDNA template (synthesized from total RNA) and primers specific for your gene of interest. When analyzing data, the expression level of the gene of interest is calculated as the target-to-GAPD signal intensity ratio. These ratios are then compared between experiments to obtain accurate relative expression values.

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. Mouse GAPD Internal Normalizer primer mix, one tube Storage Conditions: The kit is shipped at ambient temperature. Store the entire kit at -20 ºC.

Brief Protocol

For each 25-µl PCR, mix the following components in a PCR tube: 1-2 µl of a completed reverse transcription reaction using SABiosciences' ReactionReady™ First Strand cDNA Synthesis Kit (C-01) 1.0 µl of GAPD Internal Normalizer Primer Mix 2.0 µl Gene-Specific Primer Mix, final concentration of 0.2 to 1.0 µM 2.5 µl dNTPs 2.5 µl 10X Polymerase Buffer 1.0 µl Taq DNA Polymerase (Promega Catalog # M2661) Bring to final volume of 25 µl using ddH2O. Place tubes in thermal cycler. Enter and run the following program: 94 ºC, 5 min; 15 to 45 cycles* of (94 ºC, 30 sec; 50 ºC, 30 sec; and 72 ºC, 45 sec) * The PCR cycle number should be optimized for each experiment. Try using 30 cycles at first. When the PCR is complete, add gel-loading dye to the reactions and load 10 µl of the reaction onto a 2% agarose gel containing 0.5 mg/ml ethidium bromide in 1X TAE. Electrophorese in 1X TAE at 90V for 40 minutes or before the tracking dye runs off the gel. The mouse GAPD PCR product is 436 bp. Capture an image of the gel on a UV Trans Illuminator using a CCD camera or high-speed film. Acquire raw expression data: Divide the background-corrected intensity data for the gene of interest by the corresponding GAPD data from the same gel lane.