LightCycler® 480 SYBR Green I Master

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

Quantitative Real-Time PCR
The DNA standard consisted of a pGEM-T (Promega) plasmid containing a 104 bp fragment of the human mitochondrial gene NADH dehydrogenase 1 (MT-ND1) as insert. This plasmid was purified using the Plasmid Midi Kit (Qiagen) according to the manufacturer’s instructions. The final concentration of the standard plasmid was estimated spectophotometrically by averaging three replicate A260 absorbance determinations.

This DNA fragment was amplified for quantification by the high amplification efficiency (HE) primer pair (forward ND1F2∶5′-ACGCCATAAAACTCTTCACCAAAG-3′ and reverse ND2∶5′-TAGTAGAAGAGCGATGGTGAGAGCTA-3′) and low amplification efficiency (LE) primer pair (forward ND1F5∶5′-ATAAAACTCTTCACCAAAGAG-3′ and reverse ND2∶5′-TAGTAGAAGAGCGATGGTGAGAGCTA-3′).

Real-time PCR amplifications were conducted using LightCycler® 480 SYBR Green I Master (Roche) according to the manufacturer’s instructions, with 500 nM primers and a variable amount of DNA standard in a 20 µl final reaction volume. Thermocycling was conducted using a LightCycler® 480 (Roche) initiated by a 10 min incubation at 95°C, followed by 40 cycles (95°C for 5 s; 60°C for 5 s; 72°C for 20 s) with a single fluorescent reading taken at the end of each cycle. Each reaction combination, was performed in 4–6 replicates and all the runs were completed with a melt curve analysis to confirm the specificity of amplification and lack of primer dimers.

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Manufacturer protocol

Download the product protocol from Roche Lifesciences for LightCycler® 480 SYBR Green I Master below.

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