Probe FAM & VIC concentrations

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yael
yael's picture
Probe FAM & VIC concentrations

 Hi,

Can you please help me as I made a stupid mistake? I have duplex TaqMan qPCR assay. I purchased FAM & VIC probes for my duplex assay. Unite size was 6000 pmol for each probe. When I received my probes, I've completely forgotten to create stock solutions of 100µM in x1TE. I've seen on the sheet unit size 6000 pmol; volume 60µl. Therefore, I've considered my probes as already prepared stocks of 100 µM. I went from there and prepared my working solutions to be 5 µM (5ul of 6000pmol probe and 95 ul of sterile MilliQ water).

I've stupidly aliquoted 5ul of 6000 pmol of each probe into tubes and stored at -20C.
What to do from here? How can I test if a screwed concentration of probes have impact on final number of copies of target and internal control. I still have aliquots of  5ul of 6000 pmol of each probe in the freezer.
I am half way through project and had November as deadline for results delivery and limited money on the project account.
Your help is much appreciated.
Thanks.
Yaelle 
 

Brad Best
Brad Best's picture
I'm not understanding what

I'm not understanding what your problem is.  What volume did the probes actually come in?  Were your probes at 100uM, or what?  What exactly is "5uL of 6000pmol"?  Do you mean 6000pmol in 5uL - 1.2mM - or what?  It's hard to make suggestions without knowing exactly what you're dealing with.

Brad Best
Brad Best's picture
Seems I can't edit my

Seems I can't edit my previous reply.

Anyway, sorry if I seemed rude.  I was just trying to puzzle out your situation.  Did your probes come in 5uL?  So you're using 12x the concentration you thought?

The way to test it out is to do a dilution series, starting with your current 12x concentration and working down to your desired 1x concentration.  Use those to do qPCR on the same sample and see how they compare.  I suppose you could just run the 12x vs the 1x and see how it goes.  Being halfway through a study, it's hard to decide what to do.  I guess it would be best to continue using the same conditions for all samples, but probes are expensive and 12x is vast excess.

Also, are you doing ddCt?  Don't just check the Ct values, but do the actual analysis to see whether or not the relative values change.

yael
yael's picture
 Sorry guys for not being

 Sorry guys for not being clear. I've attached a sheet with a probe discription. I needed to prepare a stock of VIC with final conc. 100uM. I thought 6000pmols/60ul is 100uM final concentration so, I didn't prepare stock of 100uM.
I've just aliquoted a received probe into 5ul aliquotes to prepare 5uM working solution for qPCR. qPCR works well.

Hope this helps. My appologies for confusion.

Ilook forwardto hearing from you. Thanks.

Tijana 

yael
yael's picture
 Sorry guys for not being

 Sorry guys for not being clear. I've attached a sheet with a probe discription. I needed to prepare a stock of VIC with final conc. 100uM. I thought 6000pmols/60ul is 100uM final concentration so, I didn't prepare stock of 100uM.
I've just aliquoted a received probe into 5ul aliquotes to prepare 5uM working solution for qPCR. qPCR works well.

Hope this helps. My appologies for confusion.

Ilook forwardto hearing from you. Thanks.

Tijana 

yael
yael's picture
 Sorry guys for not being

 Sorry guys for not being clear. I've attached a sheet with a probe discription. I needed to prepare a stock of VIC with final conc. 100uM. I thought 6000pmols/60ul is 100uM final concentration so, I didn't prepare stock of 100uM.
I've just aliquoted a received probe into 5ul aliquotes to prepare 5uM working solution for qPCR. qPCR works well.

Hope this helps. My appologies for confusion.

Ilook forwardto hearing from you. Thanks.

Tijana 

yael
yael's picture
 Hi I-R Scientist,

 Hi I-R Scientist,

Thanks a lot for your reply. Just to make things clear. PRobe came as (please see sheet):
1. unit 6000pmoles
2. vol 60ul

I thought no need to dilute probe as it is already diluted in x1TE (it is how I understood froma sheet).

I aliquoted received probe into 5ul aliquotes and I used aliquote to prepare working solution (final conc. 5uM).

I am not sure if I made mistake.

HELP!!!! Thanks

Yaelle

Brad Best
Brad Best's picture
6000pmol in 60uL works out to

6000pmol in 60uL works out to be 100uM, and 5uL at 100uM into 100uL total gives you 5uM.

Crisis averted, no need to panic.

yael
yael's picture
 Thanks I-R-Scientist!!! Much

 Thanks I-R-Scientist!!! Much appreciated!