Too high CT values with TaqMan - (Dec/05/2011 )

Please Help,
I am using MagNA Pure Compact to extract viral DNA (CMV).
I used 200ul and eluted in 100ul
Then I used the LC480II for amplification using 2 different published assays of CMV. I used the LC480 probe master and HPLC primers and probes .
We quantify the viral load according to commercial standards (advanced biothechnologies) and also used Natrol CMV virus (Zeptometrix) to check the extraction
We have a problem with the quantification results. All the results are ~10 fold higher than expected according to Natrol and another lab that use MPLC instead of MPC.
THe CT values that I get for the standrds on the curve are really high: ct 27 for 10e4 copies/rxn. It does not seem right. The lab we comapre ourselves to has a rotor gene and they get ct 21 for 10e4 copies/rxn. All my samples run between ct 25-36 it looks too high. I tried playing with primers concentration and with DNA quantitiy. Would love some suggestions.

Maja25

Hi there, Lets see if I can offer some constructive comments.

Blast your primers on NCBI website and see the product length. Generally for realtime PCR, products greater than 200bp are not properly amplified and you get higher Cts. This is one problem that I have faced.

Secondly, is it possible for you to get the master mix from someone else and check? I mean, maybe the Taq mix you are using has gone bad? It is quite possible if you are getting higher Cts for different genes.

Third...what do the amplification plots look like? Even at a Ct of 27, is there a sharp rise in the curve or does the graph plateau quickly?

Finally, maybe you should check you setup for inhibitors of PCR.

I really hope you manage to solve your problem soon. Good Luck!