Parameter experiments for real-time PCR
Parameter experiments for real-time PCR
LUX primers are labeled with FAM (6-carboxyfluorescein) or JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein). In reactions without a specific template, LUX primers can quantify 100 copies or less of the target gene, and quantification can range from less than 100 to 107 copies. Using FAM- and JOE-labeled primers, the provided assay platform allows for simultaneous detection of both genes. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Parameter experiments for real-time PCR
Materials and Instruments
LUX primers Single- or double-stranded DNA or cDNA Mg2+ dNTP Hot Start Enzyme Move I. Methodology For more product details, please visit Aladdin Scientific website.
PCR Instrument
1. Primers
Primers for real-time PCR must be gene specific and capable of shorter fragment amplification.
a. LUX Primer Designer (LuxDsigner) The default parameters have been set to amplify fragments of 75~200bp length, and the primer annealing temperature ranges from 60~68°C. When setting up the PCR program, an annealing temperature of 55~64°C is more appropriate. The parameters set by the software can minimize the possibility of primer complementarity or inter-primer dimer formation. Primer design using this software is very flexible and can be designed to span the marginal or binding regions between exons.
b. Normally PCR reactions are performed with excessive primer concentrations. When using LUX primers, it is recommended that the primer concentration be 200nmol/L. Primer concentrations between 50 and 500nmol/L generally give good results. Especially for multiplexed amplification of housekeeping genes, it is recommended to use a limited primer concentration to obtain the desired results.
2. Template
The template for real-time PCR can be single-stranded or double-stranded DNA, cDNA, or circular DNA (e.g. plasmid). The template concentration is usually 10~107 copies or 1pg~10ug.
a. For real-time PCR, if the template is total RNA or mRNA, the concentration is usually 1pg~100ng. b. For real-time PCR, if the template is total RNA or mRNA, the concentration is usually 1pg~100ng.
b. The purity and integrity of RNA has a direct impact on the results.
c. RNAase and genomic DNA contamination are the most common problems, and attention should be paid to these problems when purifying RNA.
d. The presence of RNAase will degrade the RNA template, resulting in an underestimation of the abundance of expression of the target gene, while the opposite is true for DNA contamination.
e. The effects of DNA contamination can be avoided by designing primers with sequences that span the binding region between exons. However, this strategy is not always effective and it is recommended that DNaseI be added to the RNA for purification.
3. Concentration of Mg2+ and dNTP
a. Mg2+ concentrations from 1 to 10 mmol/L are ideal for certain target gene and primer concentrations, and 3 mmol/L is recommended. b. For multiplex reactions, increase the Mg2+ concentration to 5 mmol/L. c. Mg2+ concentrations from 1 to 10 mmol/L are recommended. d. Mg2+ concentrations from 1 to 10 mmol/L are recommended. e. Mg2+ concentrations from 1 to 10 mmol/L are recommended. f. Mg2+ concentrations from 3 to 10 mmol/L are recommended.
b. The ideal concentration of dNTP (dATP/dCTP/dGTP/dTTP) is 200 umol/L. If dUTP is substituted for dTTP, the concentration of dUTP should be increased to 400 umol/L. For multiplex reactions, increase the dNTP concentration to 200 umol/L.
Increase the dNTP concentration to 400umol/L for the multiplex reaction.
4. Enzymes
Hot-start enzymes are recommended.
a. In addition to templates and primers, PCR reaction mixtures are readily available from the company. For real-time PCR, PlatinumQuantitativePCRSuperMix-UDG (Invitrogen, 11720-025, 500 Reactions) is recommended; for one-step RT-PCR, ThermoScriptOne~Step-System (Invitrogen, 11731-023, 500 Reactions) is recommended. Invitrogen, 11731-023, 500 Reactions).
b. For multiplexed reactions, 3U of enzyme can be added to 50ul of reaction system to avoid lowering the efficiency of PCR.
5. Instrumentation
Real-time PCR can be performed with a number of specialized devices, as described in the user's guide. The following is a brief description of how to use the ABIPRISM7700 Model Series.