Hi guys!
Recently I encountered a problem about the Albumin (ALB) standard curve which was used for normalizing my samples.
The ALB standard curve was constructed by serial dilutions of plasmid (10^6 to 10^0 copies, 10-fold diluted). Previously, the standard curve had been done many times, and quantitative range and sensitivity (i.e. performance) were good, which could be down to 10 copies and 1 copy, respectively.
However, recently, the performance turned to be bad, with △Ct of some 10-fold dilutions being > 4.0 (criteria: 2.6-4.0), and sometimes, △Ct of replicates for some dilution points being > 1.5 (criteria: =1000 copies or even failed.
I have tried to optimize the conditions, i.e. primers & probe newly synthesized, plasmid newly cloned & extracted, or even changed another brand of PCR master mix. While, still no improvement.
Could you please give me any suggestions?
Thanks very much!
Where did you store the prepared standard? Did you make fresh dilutions to make standards each time you perform the PCR?
What kit did you use to prepare your plasmid? Which spectrometer did you use to quantify your plasmid? What was the purity of your plasmid?
Hi, Vurayai Ruhanya
Thanks for your Qs.
I made dilutions each time freshly, and the stock plasmid was stored at -30c. I used PureYield™ Plasmid Miniprep (Promega) to extract plasmid, Nanodrop to measure concentration and purity. A260/280 is 1.85, A260/230 is 1.98.
Firstly, I recommend that you should make make several aliquots of your stock. There is a danger of DNA degradation when you freeze and thaw your stock. Have single aliquot use when making your standard curves. If there is freeze and thaw several times , your standard curve quality would not be consistent
- Badges
- Science method