SYBR Green I for Real Time PCR, 100x

This plan only provides a guide, please modify it to meet your specific needs.

1. Before use, mix the product and dilute it with DMSO or ultrapure water. Usually, the product can be diluted to 20X for later use.

2. Refer to the table below to set up the qPCR reaction system at room temperature or on an ice bath (taking a 96-well plate as an example):

Reagent Final Concentration Volume for One qPCR Reaction (20μL)

10X PCR Buffer (with Mg²⁺) 1X 2μL

dNTP (2.5mM each) 0.2mM each 1.6μL

Template DNA 1-10ng cDNA x μL

SYBR Green I (20X) 0.2X-1X 0.2-1μL

Forward and Reverse Primer Mix (2.5μM each) 0.25μM 2μL

Taq DNA Polymerase (5U/μL) 0.5U/20μL 0.1μL

Ultrapure Water - To 20μL

Note:

(1) When using SYBR Green I for qPCR reaction, the Mg²⁺ concentration should be 0.5-3mM higher than that of ordinary PCR reaction without SYBR Green I.

(2) The amount of DNA template is usually 1-10ng cDNA or 10-100ng genomic DNA as a reference amount. Because the number of target gene copies contained in the templates of different species is different, if necessary, the template can be diluted in a gradient to determine the optimal template usage. When the cDNA obtained from the RT-PCR reaction is directly used as a template, its addition amount should not exceed 10% of the total PCR reaction volume.

(3) When using SYBR Green I, its usage concentration should be adjusted according to the actual situation. The optimal usage concentration of SYBR Green I can be explored within the range of 0.2X-1X.

3.qPCR reaction program: Please set up the qPCR reaction program according to the amplification efficiency of qPCR and Taq enzyme.

4. For the use of nucleic acid agarose gel electrophoresis, please refer to the following methods.

Method (1): Add SYBR Green I to the sample and then perform electrophoresis.

Mix 100X SYBR Green I working solution with DNA sample or DNA marker in a 1:1 ratio, add 6X Loading Buffer (if the DNA marker already contains Loading Buffer, it is not necessary to add it), and let it stand at room temperature for 10min. Load the sample and perform electrophoresis in the usual way.

Method (2): Stain the agarose gel after electrophoresis.

Add SYBR Green (100X) at a ratio of 4mL SYBR Green I per 100mL buffer (TAE, TBE or TE, pH7.5-8.0) (25:1) to prepare SYBR Green I staining solution. Place the agarose gel after electrophoresis in an appropriate container and add an appropriate amount of the above staining solution to at least cover the gel. Stain for 30-60min on a shaker (30-50rpm). The staining time depends on the thickness of the gel. After staining, it can be excited by blue light with a wavelength of about 500nm or detected by a corresponding gel imaging system. The staining solution can be reused about 4 times. After staining, it can be properly washed with ultrapure water to reduce background fluorescence.

Note:

(1) The concentration of SYBR Green I can be appropriately adjusted according to the actual staining effect.

(2) The effect of method (2) is relatively better, but the overall staining effect of SYBR Green I is average, especially for small fragments below 1000bp. For detection under blue light, Gel-Green (EB upgraded product, 10000X) (GK20003) is more recommended for better results.

References:

[1] Fairfax M R, Salimnia H. Quantitative PCR: an introduction[M]//Molecular Diagnostics. Academic Press, 2010: 3-14.

[2] Xiang D, Zhai K, Xiang W, et al. Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I[J]. Talanta, 2014, 129: 249-253.