Real-time PCR experiment
Real-time PCR experiment
This method utilizes the PlatinumQuantitativePCRSuperMix-UDG. When performing real-time PCR, there should be a set of equipment that can detect the fluorescence signals released during each PCR cycle. It should also be able to detect the 520mn and 550nm emission waves released after FAM and JOE excitation, respectively. This experiment is from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Real-time PCR experiment
Materials and Instruments
PCR Super Mix-UDG ROX Dye Distilled Water Reverse Primer Forward Primer Move I. Materials For more product details, please visit Aladdin Scientific website.
ABIPRISM7700 Model Series Assay Systems
1. Buffers, solutions and reagents
Take 50ul reaction system as an example.
Platinum Quantitative PCR Super Mix-UDG (Invitrogen11730-025) 25ul
ROX dye (Invitrogen12223012) 1ul
Sterilized distilled water (Gibco 15230-162) 12ul
10umol/L reverse primer 1ul
10umol/L forward primer 1ul
Subtotal 40ul
Template dissolved in 0.1XTE or sterile water 10ul
Total 50ul
2. Specialized instruments
ABIPRISM7700 model series detection system.
II. METHODS
1. PCR temperature cycling program
a. Three-step cycling method
50°C, hold for 2 min (digest UDG).
95°C, hold for 2 min (denature template; inactivate uracil DNA glycosylase; activate hot start: Taq polymerase).
95°C, 15s; 55°C, 30s; 72°C, 30s, for 45 cycles.
b. Two-Step Cycling Method
50°C, hold for 2 min (digest UDG).
95°C, hold for 2 min (denatures template; inactivates uracil DNA glycosylase; activates hot-start Taq polymerase)
95°C for 15 s; 60°C for 30 s for 45 cycles.
2. Unstranding curve analysis procedure
a. Procedure for PCR reaction: 1 min at 40°C, 19 min at 95°C, 2 min incubation at 25°C. Many real-time PCR instruments are equipped with software that provides derivative curves of fluorescence intensity versus unlinking temperature.
b. Considering the ionic environment of the reaction system, it is desirable to have only one peak in the PCR of the target product. If more than one product is amplified and/or a primer dimer is produced, multiple peaks will be generated, and of course the unlinking temperature of the primer dimer is usually lower than that of the PCR destination product. Therefore, unstranding curve analysis can be used to quickly and efficiently control the process of quantitative PCR in a very good way.
c. The results of real-time PCR using c-myc-specific LUX primers (Table 15-1) are shown in Fig. 15-2 [(a) See color figure on cover 2]. For real-time quantitative PCR, the c-myc plasmid was first diluted in a series of reagents from the range of 10 to 107 copies as described above, and the reagents were mixed according to the system described above, using the SuperMix-UDG (2 Byakuya reaction mixture). The results showed that real-time quantitative PCR using the LUX platform could amplify 100 or fewer copies of the target gene, and a dynamic curve of 7 orders of magnitude could be obtained. 
