Question

You have a sample of ssDNA molecule that you will use as a primer for PCR....

You have a sample of ssDNA molecule that you will use as a primer for PCR. You dissolved the dried ssDNA peller in TE to a total volume of 400 ul to make a "stock DNA sample." You need to determine the concentration (ug/ul) of this primer DNA in solution. To do this you took 4 ul from the "stock DNA sample" and added these to 996 ul of TE buffer in a cuvette, making a total of 1000 ul in a cuvette. Next, you determined the absorbance of 260 nm light for this 1 ml of diluted DNA in the cuvette and the A260 value is 0.014.

1) what is the concentration of the DNA solution in the cuvette?

2) what is the concentration of the primer DNA in the original Stock DNA sample?

3) what is the total amount (ug) of DNA in the stock DNA sample tube?

4) how many ul of the original "stock DNA sample" would you need to use in a PCR experiment that required a total of 0.25 ug of DNA from this sample?

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Answer #1

1) what is the concentration of the DNA solution in the cuvette?

An OD of 1 at A260 corresponds 40 ug/ml for single-stranded DNA

0.014 OD---->40 x 0.014 =0.56ug/mL

2) what is the concentration of the primer DNA in the original Stock DNA sample?

Since the dilution factor is = (4 + 996)/4 =250

Concentration of stock DNA sample = 0.56 x 250 = 140 ug/mL = 140ug/1000uL = 0.14ug/uL

3) what is the total amount (ug) of DNA in the stock DNA sample tube?

Stock DNA sample has 400uL volume with concentration 0.14ug/uL

The total DNA is = 400 x 0.14 ug= 56ug

4) how many ul of the original "stock DNA sample" would you need to use in a PCR experiment that required a total of 0.25 ug of DNA from this sample?

Required DNA= 0.25 ug

Concentration of stock DNA sample= 0.14ug/uL

= 0.25/0.14

=0.8uL

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