Question

A lab group needs to determine the concentration of DNA in their PCR product. The 500bp band of the SS (DNA Size Standard) on a gel contains 50ng of DNA. The lab group loaded 10ul of PCR product + 5ul of Blue Juice Dye which resulted in a band 3 times as bright as DNA SS. A.) How many ng of DNA is in their PCR product? B.) What is the concentration of their PCR product (ng/ul)? C.) If the lab group needs a final concentration of 3-10ng/ul for sequencing, will they have to dilute their sample? Please show work and explanations.

Answer 1

The 500 bp band on a gel contains 50ng of DNA.

Now the intensity of the band with the PCR product is 3 times to that of the standard SS DNA(50 ng)

Therefore, Amount of DNA in the band formed by loading 10 ul of PCR product is = (3X50)ng =150 ng.

The concentration of the PCR sample is = amount of DNA/Volume of PCR product= (150/10) ng/ul = 15 ng/ul

As this PCR concentration is much higher than than the PCR concentration required for sequencing, it needs to be diluted with TE buffer.

[Amount of TE buffer needs to be added to make it 10ng/ul is ((15/10)-1)= 0.5 ul]