How can I prepare standard DNA for QPCR - (Sep/01/2006 )
Currently I am working on insect DNA using PCR. I would like continue on QPCR and for this I would like to know about the standard DNA samples protocol.
What in particular do you want to know?
Are you planning to do real time PCR with genomic DNA or with cDNA?
Do you already have primers and probes that you are planning to use?
Will you use SYBR green or a specific probe?
Is the gene of interest expressed at a high or low level?
Hi , I've started working with qPCR last week(before that I had done only 1 PCR, quite a change). I'm using cDNA as templates, a different dilutions. The first time I run the different dilutions I'v got a superb standard curve. BUt when I try to replicate it it never works, I'm far from the concentrations of copies I'm supposed to have. What would you suggest. I though I should redo my dilutions everytime I run the q PCR so they will be fresh with NO contamination. I'm using Rotorgene 3000 with Sybr.
How to prepare you rstandard....
You may run a PCR using your target specific primer--> clean up your PCR product--> quantitate your PCR product using spectrophotometer--> convert your spectro reading into DNA copy number--> perform a 10 fold serail dilution on you standard and subject to real-time PCR.
You should a able to observe the amplification curve comming up sequencailly interval by 3.3 cycle :-)
since your starting meterial is cDNA while you standard is dsDNA, you should perform a 2nd strand DNA synthesis prio to your PCR.