"Undetermined" Ct, but product IS formed?!?! - (Sep/14/2006 )
After not getting any Ct or increase in abs. after my qPCR run (all of row B and E failed), I decided to run the reaction out on a gel. When I look at the gel, there is a clear, very distinct band in each lane (which is the expected product)!! What's going on?!?! Any suggestions? btw I'm using a 7900HT ABI, and using automatic threshold/Ct function in SDS2.1
Cheers,
Dave
did you run a melting curve analysis? if so, what did it tell you?
hmmmmm have you had your machine serviced lately? is there an issue with the bulbs or the computer connection hardware?
Cheers,
Dave
TaqMan or SYBR green?
It is SYBR green. I'm running a new gel now with no EtBr to see if I can still see the bands. That is the only thing that I can think of.
I checked with the person in charge of the machine, they said that it has been working perfedtly recently. I did not do a melting curve. Should I do this routinely?
if you use SYBR green, you must run a melting curve every time
could you please post a pic of your gel?
could you please post a pic of your gel?
Ok, in future I will always run the melting curve, although a post doc in my lab said that running a gel after gives you more info (band size). I've finished running the gel with no EtBr staining (using the SYBR green as a stain instead) - and there is a clear band in the "undetermined" wells - ruling out lack of SYBR in one of my master mixes. I'm attaching a jpg of one of my gels too. You can see product for all genes (except 2 lanes of gene 2) - "Undetermined" is gene 1.
I was hoping for size
are you absolutely CERTAIN you're not getting primer-dimer?
as far as the melting curve...any old DNA band of approx the same size can masquerade as your product. with a melting curve, I think you can get a bit more specific. you can determine for certain that you have no gDNA contamination, and you can rule out primer-dimer or mispriming events. I would not say that a gel is better information. personally, I only run a gel if there is a problem, otherwise everthing I get is from the software on the machine.
the whole point to real time is that you don't have to muck around with gels
are you absolutely CERTAIN you're not getting primer-dimer?
I'm unsure what you mean about size? Do you mean the size of the product? Also, I"m not sure that I don't get primer-dimer, although the same primers have been used for regular RT-PCR and I did qPCR using these primers for the first time last week, with no apparent problems. I think the best thing for me to do is re-run the qPCR and see if I get the same results.
Thanks for your help, Aimikins!
well, as far as the size...depending on how you set up your assay, usually products are in the 100-150bp range. primer-dimers usually show up in the 40-70bp range. of course, this is not always the case, but often. so, if your bands that yielded no product are smaller than the product that you were hoping to get, it could be that primer-dimers are forming. this reaction competes with the reaction for product and can result in so much primer-dimer that no product is seen
if you ran the assay before and it worked correctly, but not the second time...what are the chances that an error was made when diluting/adding from your primer stock on the second run? for example, if you were off by a factor of 10 and added way too much primer, then perhaps you favored the primer-dimer reaction in the second run and not the product-forming reaction