Question

Results for QPCR report:

Absolute quantification (Standard curve) – Using a dilution series of known template controls – Fluorescence is measured against the standard curve – At least four points (in duplicate) should be run for an accurate curve to be produced – The linearity of the curve (Rsq value) should be close to 1

• To indicate the amplification efficiency is consistent across all template concentrations

Your DNA standard 1 contains 1.0 x 106 copies of DNA, and you will use this to make your dilution series. You are required to each make 10 fold dilution series:

tube 1: 10^6 copies, tube 2: 10^5 copies, tube 3: 10^4 copies and tube 4: 10^3 copies

Standards				Samples
Copies per uL	Log copies per ul	CT value		Name	Cq value
10,000,000	7	27.39		Case	35.56
1,000,000	6	30.43		Positive	26.05
100,000	5	34.29		Ext Neg	44.33
10,000	4	37.38		PCR Neg	N/A

The Cts as provided are correct and have been verified. I'll confirm that there is a single melt curve peak in each of the samples, all of the same size.

Discussion: (~ 1 paragraph)

• State your key findings?
• Were the results accurate?
• Were you able to quantify the DNA on the swab?

Interpret and discuss the quality of your data.

If your standards behave 'quantitatively', then there should be a 3.3 cycle shift between each 1/10 dilution. You can work out how 'off' your standards are by working out your dilution factor between each successive standard (e.g. if you got a 4.3 cycle shift, then you would have diluted the sample 2^4.3 or ~1/20). Note the cycle shift between each subsequent pairs of standards will be different.

Other things to consider: 1. if you don't see a 3.3 cycle shift what sorts of thing could cause error in this aspect. 2. Do you think this is an accurate method of quantification.

Need help with discussion part, Thanks.

Case swab CT (y) = 35.56

Y=-3.383x + 50.979

35.56=-3.383x + 50.979

35.56 -50.979 = -3.383

-15.4 / -3.383 = x

X = 4.55

Answer 1

Since there is a single Melt curve it involves DNA has double-stranded PCR products to a gradual increase in temperature.

The dsDNA issociate as the temperature has increased

And so in results ther will be sharp decrease in fluorescence as the probe can no longer bind to the PCR product.

melting temperatures may be PCR product specific

it is dependent upon the length of the product and the type of nucleotides present.

Melt curve analysis can often be implemented following qPCR on the same instrument, improving the discriminatory power of the assay.

Assay efficiency and repeatability can be predicted from the line of best fit: amplicon accumulation is proportional to 2n , where n is the number of amplification cycle repeats. Therefore:

2 n = fold dilution,

- 2 fold dilution n~1,

- 10 fold dilution n~3.323

Therefore when a 10 fold serial dilution is performed, the amplification plots for each dilution should be 3.3 cycles apart.

Quantification strategies: Standard curve quantification versus comparative quantification

For standard curve quantification, the curve is constructed using a template containing a defined copy number of template molecules, most usually an artificial oligonucleotide or linear plasmid containing a clone of the target.

Standard curve quantification can also utilise template material of unknown concentration, such as a preparation of cDNA or gDNA.

So it is not correct method of quantification.