I'm a first year graduate student from University of New Hampshire (started my MS program at Fall '08) and dove into using qPCR to detect Vibrio parahaemolyticus and Vibrio vulnificus within oyster homogenate (the basis of the project and the crucial portion of my thesis).
Now I've been using a gDNA standards for a while and when i start to run out of my 100ng stock I make a new genomic prep (CTAB/Chloroform extraction) from an overnight culture of Vp and Vv. Now my committee member (of the lab that has the real-time machine I work in) told me that it would be a good idea to create aliquots of 100ng of gDNA and store them in the -20C until I run out of my working stock. So I did that and once I ran out of my working stock, I defrosted the 100ng of gDNA, specced it to make sure the concentration was correct (which it was) and diluted to 1fg (10-8).
Now before I froze my gDNA and just remade preps from o/n cultures, I had PCR efficiencies that ranged from 98% to 108%. I noticed that my PCR efficiency dropped to about 85%, and since I've been using this working stock for about a month or so (I kept a 100ng working stock in the fridge that I've been using to re-dilute the standards and this has been in my box for about 2 months) I decided to change my gDNA (since it's easy to remake new stocks).
Now after running a qPCR with the newly aliquoted frozen gDNA my PCR efficiency has dropped to 70% - 85%. which is a huge change and I am not sure why? Is it possible that freezing the DNA and them thawing them out affect the PCR efficiency that much? I know that there are many different factors that can affect the efficiency of PCR but I'm not doing anything different.
I am currently running a Taqman assay and the probe I was using was newly thawed aliquot and I used this probe on the old working stock of gDNA as well as the new working stock of DNA. I'm also using a pre-made mastermix (IQ Supermix from Bio-Rad and adding additional Mg2+ to make the concentration of Mg2+ to 5mM) and the primer stock I've been using have been freeze/thawed 5 times (which also I've never had trouble with in the past).
I'm going to run another standard on Sunday with new frozen aliquot of primers to see if these primers can raise the efficiency but I'm just wondering if that freezing the DNA before using it affects the efficiency?
(If this is a dumb question I'm sorry, when I got this project, I've never done a real-time in my entire undergrad research career [during that time I was doing more cell culture and cytotoxicity assays]).
Any input would be great thank you.
These are the rest of my PCR reaction related information:
1 x IQ Supermix
*2mM of Mg2+ (To make it a total of 5mM of Mg)
*150nm of Probe
*75nm of Primers
(I also went from 25ul rxn to 15ul rxn to save some reagents would THAT affect the efficiency as well, although the first reaction I ran with the new gDNA was 25ul and it gave me an efficiency of 82%)
*These are the concentrations that I'm using that is from a published protocol by Nordstrom et al. 2004 but instead of using the smartcycler I am using the Icycler and running MyIQ for the program that came with the machine*
95C - 3 minutes (for the IQ Supermix)
*95C - 15sec
*59C - 15sec
Thank you for your time and sorry if this is a nooby question,
Freezing DNA have effects on real-time PCR efficiencies
1 reply to this topic
Posted 20 July 2009 - 01:14 PM
I've been taught not to freeze genomic DNA, but keep it at 4C, because freezing will cause it to shear. So, the concentration after thawing would be the same as before it went into the freezer, but the DNA will have been broken up. I don't know if that would cause a change in your efficiencies or not, but the freezing may be affecting your gDNA template.