Typical results from one of our Kapa Sybr Fast qPCR runs on a Bio-Rad CFX System.
Info: View Product View Product Specs
In our lab, we perform a great deal of qPCR on DNA isolated from mouse stool samples. The purpose being to determine intestinal micro flora changes in the setting of our experiment. Speed and cost are of great importance. The Kapa SYBR Fast is a standard 2x SYBR Green qPCR master mix that has the added advantage of significantly reducing run time as well as cost per sample compared to other qPCR master mixes available.
Microbe detection using qPCR
We collect a single stool sample from each mouse. We then extract DNA from the stool sample using a Qiagen Stool DNA Mini kit run on the QIAcube.
With the extracted mouse stool genomic DNA, we then run qPCR reactions for various microflora using the following reaction set up (per 20uL reacton): 10 uL Kapa Sybr Fast 2x + 2uL primers + 4 uL DEPC H2O + 4 uL sample DNA. This is then run on a Bio-Rad CFX Real-time PCR System, along with standards for each target. The protocol we use is as follows: 95°C 5 min, [95°C 10 sec, 61.5°C 30 sec ] x 40 cycles, followed by a meltcurve protocol.
Control ROX was included with this kit. I was using a Bio-Rad CFX that does not require the control/calibrating dyes. However, it is important to be aware of the calibration requirements for your particular qPCR system.
The figure below shows the results of a typical reaction. As indicated, the standard curves are always very accurate and our samples come up very quickly and consistently.
Any PCR run that is done is only as good as the material that is used. It is critical to use good sample isolation techniques and select primers that are specific and accurate.
Product Name: KAPA SYBR® FAST Universal 2X qPCR Master MixSupplier Page