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absolute quantification


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4 replies to this topic

#1 chn09

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Posted 23 May 2010 - 03:52 AM

Hello everyone
Is is necessary to clean up the linearized plasmid that is to be used as template in real time PCR. And how to convert primer concentration of 10 micromoles to 900 nanomoles?
pls help

#2 vladooo

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Posted 23 May 2010 - 12:07 PM

1. Yes, clean-up.
2. 10 micromoles = 10 000 nanomoles => 900 nM = 0.9 uM.

#3 sssbio

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Posted 29 May 2010 - 02:00 PM

Hi,

Will you please guide me to understand what if I have no choice of housekeeping genes to be used to normalize real time PCR to quantify mRNA expression levels? I tried all possible housekeeping genes, however, they all were significantly showed down regulation during experiment. Is there any alternative method that will help me to quantify mRNA expression levels of targeted genes?

looking forward for suggestions

sincerely

sss

#4 chn09

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Posted 30 May 2010 - 09:10 PM

hi ssbio,
Are you working with a prokaryotic/ eukaryotic system?. If u are using a prokaryotic system, 16 s rRNA would be a good candidate for normalizing your data. Other methods for quantifying mRNA transcripts would be Northern blotting, Semi - Quantitative PCR etc.

hope it helps..

#5 sssbio

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Posted 01 June 2010 - 05:18 AM

hi ssbio,
Are you working with a prokaryotic/ eukaryotic system?. If u are using a prokaryotic system, 16 s rRNA would be a good candidate for normalizing your data. Other methods for quantifying mRNA transcripts would be Northern blotting, Semi - Quantitative PCR etc.

hope it helps..


Thanks a million for your guideline,

Today, I tried 18 S rRNA primers and it worked very well. I could see bands of same intensity on agarose gel in both control and treatment. And hoping this will work in real time PCR as well. My project is related to understand plant responses to different environmental conditions.

Thanks.




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