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DNA Quantification

Operating the Ultrospec 2000 UV/Visible Spectrophometer

1)    Dilute 2 ul extracted genomic DNA in 68 ul TE buffer or water in a 1.5 ul eppendorf tube.

2)    Turn on the Ultrospec 2000: UV/Visible Spectrophotometer. The power button is located on the back right of the machine just to the left of the chord.

3)    Press the MODE button.

4)    Press the left arrow button until you get to NUCLEIC ACID.

5)    Press up arrow button to get to DNA.

6)    Press ENTER and wait for 10 minutes for machine to warm up

7)    For background, place blinking cursor over N. Press Enter. You are now ready to load the reference sample.

8)    The reference sample is simply 70 ul of TE buffer or water (whichever you diluted your sample with). Pipette the entire reference sample into the sample cuvet. The cuvet should be located in a plastic case in the top drawer to the right of machine. Although the cuvet may look pretty busted already, be sure to handle it with care: new cuvets are expensive and this one isn’t even ours! Be sure that your sample fills the bottom of the cuvet.

9)    Place cuvet in BLUE CHAMBER of machine. When the sample is loaded, the blue chamber should be at 12:00. You will see a small square towards the base of the cuvet on two sides. Make sure that those squares are facing left and right to allow the light to pass through.

10) Press SET REF button. After reference is set, you will hear the machine move the sample one position to the right.

11) Remove cuvet, pipette out the reference sample, and discard reference sample into waste.

12) Pipette your first sample into the empty cuvet, being sure it fills the slot at the bottom of the cuvet.

13) Load cuvet into the TOP position of the machine

14) You’re now ready to run the analysis, but before doing so you should be prepared with your notebook and a pen to record the output, which will flash on the screen in timed intervals. Press RUN button, and record 260, 280 and C scores as they are displayed by machine.

15) Press the down arrow button to get the desired ratio and record this value

16) Remove cuvet from chamber and pipette out sample into waste. Rinse cuvet with TE buffer or water before repeating steps 13-16 with your next sample.

Calculation and Interpretation

1)    You are going to use the data obtained from the spec to calculate two values: (1) the concentration of DNA in your sample and (2) the purity of your genomic DNA.  The 260 score is used to calculate the DNA concentration whereas the 280 score indicates the amount of contaminants in the DNA. You want your 260 score to be higher than your 280 score.

2)    A 260/280 ratio greater than 1.5 is indicative of a very pure sample. If your ratio is much lower than 1.5, this could be indicative of high levels of protein, phenol or other contamination in your DNA.

3)    Calculate the concetration of your sample in ng/ml using the following equation: [ng/ml]= 260 x 50 x dilution factor. The dilution factor is 35 if you used 2 ul DNA in 70ul water. You want the concentration of your sample to be around 10-15 ng/ml. If it is much higher, dilute down your DNA with water prior to use.  If it is much lower, you may need to redo your extraction.

Posted by Ali Ossip-Klein – Dec. 5th, 2009