Im planning to do qPCR on E. coli K12.
I read an article which stated that dxs gene was used as housekeeping gene in E. coli. Alkaline phosphate (phoS), beta-D-glucuronidase (uidA) or acid phosphatase (phoA) can be used as housekeeping gene?
May I know any others housekeeping gene that i can use?

Hi everyone
I am doing qPCR in Corbett machine and having some problems with a really high efficiency. I’ve tried almost everything that I’ve read could cause that problem:
1. Increase the Temperature
2. Change the amount of the MgCl2, which was a slightly better, but still 130-150 % efficiency
3. Change the amount of the primers
4. Played with the dNTPs
5. Make different dilutions

By now I am using only DNA from Promega (female) and this is the only one thing that I didn’t change just because it is standard DNA and I don’t think it could be the problem. Have anyone had the same problems with the standard DNA? And do you have any ideas what could couse the problem?

Thanks

Katya

Hello,
I'm assuming there's a fairly fundamental reason why this isn't done, at least I haven't been able to find any informative reference so far...

I want to accurately quantify DNA, total RNA (~rRNA) and mRNA across many, very small samples (mutants and timecourse). DNA, easy enough, but for RNA I don't see why I can't just RT using either random or oligo(dT) primers, and then run the qPCR rxn with random primers (I have random 15mers, Tm very low at 40C though). I don't want to make ANY assumption about 'reference' mRNA here - the conditions are potentially highly dynamic. Possibly I could go for specific primers to the major rRNA species, but that would be a bit of a pain.

Anyone else tried this or know of a reason it would not work?

I've done a single trial run, 10ul rxns with 10-fold dilutions from 10ng down to 10pg, but no take-off whatsoever, nothing on the melt. I might try larger rxns, maybe 20mer primers, higher [primer]. It would be very nice if this would work, but perhaps I'm being too simplistic.

Cheers

Hi everyone
I recently started doing qPCR and have used 18srRNA and GAPDH as reference genes, looking for that one which varies less with the different treatments.

One thing I am not sure about is the degree of variability of the reference gene that is considered to be safe ( since the ideal reference gene does not exist).

For example, when I look at the fold change experienced by my reference gene when comparing CTRL versus PE treatment, I get 1.4.

Is 1.4 too much of a change or is it within the allowed range?

Thanks in advance

We are considering to have a 384-well qPCR system. There are three models out there, including ABI 7900HT, Biorads CFX384 and Roche Lightcycler 480. Which one you think is better?