You have a sample of small single-stranded DNA (ssDNA) molecule that you will us
ID: 198456 • Letter: Y
Question
You have a sample of small single-stranded DNA (ssDNA) molecule that you will use as a primer for Polymerase Chain Rection (PCR). you dissolved the dried ssDNA pellet in Tris-EDTA (TE) buffer to a total volume of 400 ul (microliters)to make the "Stock DNA sample". you now 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 spectrophotometric cuvette, making a total volume 1000 ul in a cuvette. next, you determined the Absorbance of 260 nm light (A260) for this 1 ml of diluted primer DNA in the cuvette, and A260 VALUE IS 0.014.
(i) what is the concentration (ug / ul) of the primer DNA solution in the cuvette (must show calculations)?
(ii) what is the concentration (ug / ul) of the primer DNA in the original Stock DNA sample (must show calculations)?
(iii) what is the total amount (ug) of DNA in the stock DNA sample tube (show calculations)?
(iv) how many ul (microliters) 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 (show calculations)?
Explanation / Answer
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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