real-time pcr plasmid standards - (Apr/24/2005 )
Hello,
I have been struggling to optimize two real-time PCR assays. I am using recombinant plasmids containing my gene of interest to generate my standard curve. I just recently learned that I should be linearizing my plasmids, as well as using siliconized tubes to avoid DNA sticking to the tubes. I was hoping someone could give me some pointers on how to do this. Most restriction enzyme digestion protocols recommend small volumes (10- 50 microliters). I make 1:10 serial dilutions in large final volumes (1ml). I find working with a minimum of a ml prevents variability associated with pipetting error, etc. So, basically I need to know if I can perform a restriction enzyme digestion on a larger scale??? If I can't, my plan was to perform multiple restriction enzyme digestions on aliquots of the same plasmid stock, run them in a gel, purify them from the gel, pool it all together to get a large volume, get a total DNA concentration using a fluorometer, and with this reading prepare 1:10 serial dilutions. These 1:10's will be run as standards in my real-time run. (hopefully I am explaining this clearly). I am just struggling to find a way to make my assays reproducible with little variation run to run!! Any advice welcome.
Thanks in advance fellow real-timers 
Quick question ... siliconising the tube ... is this for the PCR reaction i.e. are you using a lightcycler OR is this for storing the DNA?
STORING DNA ... most microcentrifuge tubes that are for molecular biology purposes are low adherent for DNA. If you are concerned, buy specific low binding tubes (Eppendorf and other companies have them). For siliconised tubes ... I think you can buy them.
TO LINEARISE OR NOT??? what was the reason given to linearise? I have been using plasmid DNA in PCR reactions for years and have never linearised. Usually the denaturing step if using the correct temperature will eliminate secondary structure problems.
TO LINEARISE LARGE-SCALE ... is doable. The main limitation is your method of cleanup. Gel purification is not applicable to large-scale digestion but column clean-up is i.e. microcon units and qiagen columns. You will still need to run some digest on a gel to verify complete digestion.
Hope this helps
STORING DNA ... most microcentrifuge tubes that are for molecular biology purposes are low adherent for DNA. If you are concerned, buy specific low binding tubes (Eppendorf and other companies have them). For siliconised tubes ... I think you can buy them.
TO LINEARISE OR NOT??? what was the reason given to linearise? I have been using plasmid DNA in PCR reactions for years and have never linearised. Usually the denaturing step if using the correct temperature will eliminate secondary structure problems.
TO LINEARISE LARGE-SCALE ... is doable. The main limitation is your method of cleanup. Gel purification is not applicable to large-scale digestion but column clean-up is i.e. microcon units and qiagen columns. You will still need to run some digest on a gel to verify complete digestion.
Hope this helps
We bought siliconized tubes for DNA storing to reduce DNA binding.
About linearizing the plasmids, I learned this just recently. I have been told circular or supercoiled DNA will not amplify the same efficiency as linearized. I am assuming when you say you use plasmid DNA in PCR you are referring to real-time PCR?!? I was using circular plasmids, and was having some problems with a SYBR Green assay's reproducibility and was having trouble getting a TaqMan assay up and running. Thought maybe this will help me out to start linearizing it.
Thank you for the restriction enzme digestion suggestions. Very helpful!
-Bridget
Hey Real Timers!!
I am attempting an M.Sc project regarding MRD monitoring in CML patients. We do not wish to add a cell culture section to the project in order to produce our own plasmid standards. Ipsogen only manufactures b3a2 and e1a2 BCR-ABL plasmid copy number standards. CAN ANYONE PLEASE TELL ME OF A COMPANY THAT MANUFACTURES b3a2 as well as b2a2 plasmid standards. We urgently need these standards before we can start with the RQ-PCR
Till later,
<japie> 