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Selection of RT-PCR

Selection of RT-PCR - Real-Time PCR and Quantitative PCR Forum

Selection of RT-PCR - Real-Time PCR and Quantitative PCR Forum


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  #1  
Old 07-02-2006, 03:56 AM
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Default Selection of RT-PCR



HI
which type of RT-PCR is better for experimentation if we consider economically and also efficiency of performance.
1- With SYBR green dye
2-FRET-RT-PCR

what are the advantages and disadvantages of these PCR.

Waiting for answer
aftab
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Old 07-02-2006, 07:08 PM
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Default Re: Selection of RT-PCR

SYBR green dye seems to be the standard for RT-PCR. I think FRET is quite new so I would stick with SYBR
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Old 07-20-2007, 02:45 PM
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Default Re: Selection of RT-PCR

I would also choose SYBR green dye.

The advantages of the SYBR Green dye chemistry are as follows:
• It can be used to monitor the amplification of any double-stranded DNA sequence.
• No probe is required, which reduces assay setup and running costs.

Disadvantage of SYBR Green I Dye
The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.
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Old 07-23-2007, 03:54 PM
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Default Re: Selection of RT-PCR

SYBR green can work but you must optimize so that no nonspecific products are generated---optimize using traditional PCR. Then a simple primer concentration optimization--usually reduction in primer used in traditional PCR--plus Melting point determination. You must also have a positive control reaction that works without generating nonspecific products---this control will give you a melting point (pcr amplicon's) that you can compare in your unknowns. That way if the melting points are the same---you can conclude that the SYBR green expression signal is all due to the one pcr product--and not to any primer dimer or nonspecific bands.
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