For my real time pcr assay,I am using plasmid DNA and am preparing a 10 fold dilution series ranging from 10^9 to 10^2 copy numbers.What i observe that there is amplification at high copy numbers and no amplification at low copy numbers and also the points do not fall on the linear plot.I dont understand where am i going wrong.
First major cause in cases is dilution. But as you have encountered this problem, I m sure, you must have repeated dilution and run the experiement again. Also let me know how actually you dilute the plasmid? Use higher volumes so that you meet precision e.g. From the stock of 10^9 take 20 uL DNA and add into 180 uL Nuclease free water (This makes your 10^8). Vortex and brief spin the mixture. Take 20 uL from this 10^8 dilution and add to next 180 uL of Nuclease free water. continue further.
Can you specify below so that I may give some suggestions
1) Size of amplicon and size of plasmid DNA
2) Chemisty- SYBR or Taqman
3) Temperature at which Data collection step is incorporated?
Okay....
1.Amplicon size -330bp, Plasmid Size-3150
2.Chemistry-SYBR green SSo fast
3.Annealing temperature is 62.
I dilute my plasmid dna by taking 1ul of sample and 9ul of nuclease free water.
The standard curve is very important for my assay as I will be needing this curve to quantify my samples from soil dna.
I would recommend using a series of 2-fold dilutions (not 10-fold) with non-specific carrier DNA. Two-fold dilutions are very accurate even when the pipette is inaccurate, and carrier reduces adsorption. Pipet up and down 10 times and replace tips each dilution.
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