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real time qPCR


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#1 Ribosoul

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Posted 16 March 2005 - 01:16 PM

Hi everybody :D
I use ABI7500. From the dissociation curve you know the melting point of the PCR products. I perform duplex pcr. So I have two pieks. Can anyone tell me what I am told about the heighs of the pieks?
Thank a lot : )

Ribosoul :P

#2 yongyk

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Posted 16 March 2005 - 07:49 PM

Dear Ribosoul,

The height of the piek represent Relative Fluorescence unit (RFU). It tells you qualitatively the amount of the specefic product contain in your tube.

I think the more relevent info getting from the dissasosiation curve is the specific meltting temperature. Dont't worry about the height.

Regards
Yongyk :P

#3 Ribosoul

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Posted 17 March 2005 - 10:36 AM

Dear Ribosoul,

The height of the piek represent Relative Fluorescence unit (RFU). It tells you qualitatively the amount of the specefic product contain in your tube.

I think the more relevent info getting from the dissasosiation curve is the specific meltting temperature. Dont't worry about the height.

Regards
Yongyk :)

Dear Yongyk,
Thankyou for your answer. What do you mean 'It tells you qualitatively the amount of the specefic product contain in your tube'?
Since the instensity of RFU is according to the pcr product copies, why I cannot say a pcr product with a higher piek has more copies? Here I have to emphasise I am talking about twe different pcr product IN the reaction matrix.

Ribosoul :rolleyes:

#4 yongyk

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Posted 17 March 2005 - 12:20 PM

Dear Ribosoul,

Let me clearify with you. What do you means by "more copies". Are you reffering to initail copies or DNA amount of your PCR product?

1) Ct Value tells you the information of the initial copies.

2) RFU during the amplification curve will tell you the amount of your total PCR product (specific product + primer dimer).

3) RFU during the melting curve will tell you amount of specific PCR product.

Since standard curve is build base on Ct value (initial copy) thus there is no way to tell the exact number of the end product. That why I said is "qualitative".

You can only only say a pcr product with a higher piek has more copies (end product) when both of your PCR product are in similar size. Why?

Just compare a fragment of 350bp and a fragment of 105bp. The larger one will incorperated more SYBR Green molecule than the smaller one, and end up with higher RFU.

When you build a standard curve by performing 10 fold series dilution, you will see your height of you melting peak decreesing from the high to low concentration.
when you take the PCR product and run a gel, you will see the sample with highest melting peak will have nost intense band, whereas the lowest peak you might not be able to view on gel.

Thank you

Best Regards
yongyk




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