Troubleshooting in quantifying Genomic/WGA DNA - (Jul/18/2008 )

Hello everyone!

I am trying to quantify Whole Genome Amplified DNA by Real-Time PCR using a Standard Curve experiment and SYBR Green reagents.

I made 1:10 and 1:100 dilutions from the WGA samples but the quantitation results that I get are not consistent with the dilutions. I would expect to get a 10-fold difference between both dilutions but actually I get a 2 to 5-fold difference, i.e. the samples that are 1:10 diluted only show 2-5 times more DNA than the samples that are 1:100 diluted.

What could be causing this and how should I know which are the values to be trusted?

Thanks in advance.

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Maybe your DNA is too much concentrated, so the lower dilutions are still at the reaction limit. Try more dilute samples like 1:1000.

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Thank you for your reply, trof.

It would hardly be the case, because I'm getting concentrations as low as 2ng/ul. If I dilute them more, I will probably get concentrations that fall below the high-confidence range.

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When I do qPCr on my WGA DNA we typicially use 10ng per rxn. I am surprised you are getting such a low concentration after you amplify? What kit are you using to amplify your DNA?

Clare

PS: Happy Friday!

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I'm using Genomiphi V2 from GE Healthcare. How much DNA do you get after amplification?

Thanks

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Hi,

Sorry for the late reply - I have been on holiday

We use the WGA sigma kit. Yesterday I amplified a few different samples (starting with 20ng or 40ng) and ended up with about 2.6-4.6ug.
I am not familiar with the kit you are using so can't offer any more advice on it

Clare


[/quote]

I'm using Genomiphi V2 from GE Healthcare. How much DNA do you get after amplification?

Thanks
[/quote]

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