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Real time genomic amplification-copy number assay-non specific product ????

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Posted 28 September 2010 - 12:11 PM

Hi everybody,
i am here to get some suggestion to correct my qpcr expt aiming to asses the copy number of a gene in various cell lines. first i explain what i did so far. i isolated DNA from various cell lines and got genomic targeting primers for my gene of interest from literature and started doing qpcr. i have DNA from a normal cell line as a calibrator and B-actin and another gene as an internal reference genes. I used 20ng of DNA from each cell line and did real time PCR using SYBR GREEN in icycler machine. primer concn used 4ng final conc (used 1 micro litre of 0.1 micro gram per microlitre each to final 25 microlitre reaction).

Thermal protocol,
Cycle 1: ( 1X)
Step 1: 55.0ºC for 02:00
Cycle 2: ( 1X)
Step 1: 95.0ºC for 10:00
Cycle 3: ( 40X)
Step 1: 95.0ºC for 00:30
Step 2: 60.0ºC for 01:00
Data collection enabled.
Cycle 4: ( 80X)
Step 1: 55.0ºC for 00:10
Increase setpoint temperature after cycle 2 by 0.5ºC
Melt curve data collection and analysis enabled.

since i got the primers from literature i dnt know the exact melting temperature of the primers and from some online softwares i analysed and found that the tm of the primers is around 63. (i ran two PCRs one is above and another with only change in Ta ie.,62 but both tm curves are same) the amplicon size is around 500 bp. to me the melting curve doesnt look normal, jus to check i ran a 2.5 per agarose gel and i have attached both the melting curve and gel for ur reference. pls hav a look and advise me if anything wrong in that.

Picture description,
This PCR is just to standardise the expt. i used three genes two reference and one gene to be analysed for copy number. Dna from normal cell line ..
the red color peak in melt curve is the product that was in first lane of the gel, 2nd is its NTC, third lane for the middle curve in the melt graph (which shows typical non specific amp as per the tm curve), 4th lane is its NTC, 5th lane corresponds to the big peak in the melting curve graph and 6th lane is its NTC...

Attached Thumbnails

  • amp product on gel.jpg

Attached Files

Edited by GNANA, 28 September 2010 - 12:13 PM.

I always had an alternate hypothesis....

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