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Problems with MS-HRM Standard Curve!


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#1 Bianabi

Bianabi

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Posted 02 August 2012 - 12:34 AM

Hi!
We're trying to optimize some Methylation HRM experiments with different MasterMixes, Dyes, PCR conditions, primers "home-made" and published, Tannealing etc...
We've also tried different bisulfite conversion kits, and different Methylated and Unmethylated Controls. Now we're using Cells-to-CpG Methylated gDNA Control (Applied Biosystems) for 100%Methylated and Placental gDNA for 0%Unmethylated in order to prepare a standar curve.
We use a 7900ht Fast PCR System from Applied.
Our problem is that we've got almost the same result with Standard Curve. Always!!
In the Normalized Melt Curve and Difference Plot Graphs (Software HRM from Applied), we have 25-100% methylated lines very close.
We just can differentiate 0-20% more or less.
All our experiments combinations are in order to improve the distance of high methylated standard lines.
Could you give me some idea?
Thank you and sorry for my english!
Ana




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