Question

From the UV absorption data comment on the purity of your DNA preparation.

*Not sure what extra data is required? All data given to us to complete lab is here.
234nm 0.350 260nm 0.524 1st Der. 1st Der. 0.386 0.385 64.02 Temp 55.02 57.02 59.02 61.02 63.02 65.07 67.02 69.04 71.02 73.07 74.97 76.96 79.07 80.97 83.07 85.07 87.00 89.02 91.07 93.02 95.02 Wavelength Absorbance Control Abs Temp 0.385 57.02 0.385 58.02 59.02 60.02 0.386 61.02 0.386 62.02 0.386 63.02 0.386 64.02 0.388 65.02 0.390 66.02 0.395 67.02 0.398 68.02 0.401 69.02 0.418 70.02 0.437 71.02 0.479 72.02 0.510 73.02 0.516 74.02 0.518 75.02 0.519 76.02 0.521 77.02 78.02 79.02 80.02 81.02 82.02 83.02 84.02 85.02 86.02 87.02 88.02 89.02 90.02 91.02 92.02 1.71E-04 4.93E-04 1.47E-04 -2.05E-04 1.50E-04 5.18E-04 7.00E-05 -3.54E-04 -1.54E-04 -3.50E-05 1.73E-04 3.68E-04 5.18E-04 6.62E-04 9.39E-04 1.15E-03 1.74E-03 2.43E-03 2.07E-03 1.81E-03 1.47E-03 4.73E-04 4.97E-03 9.71E-03 9.10E-03 7.92E-03 1.45E-02 2.19E-02 1.87E-02 1.67E-02 9.43E-03 2.27E-03 2.17E-03 1.09E-03 7.12E-04 4.56E-05 280nm 0.283 0.03M NaClO4 Temp Abs Temp 55.02 0.370 57.02 57.22 0.369 58.02 59.23 0.369 59.02 61.32 0.370 60.02 63.22 0.370 61.02 65.27 0.370 62.02 67.27 0.370 63.02 69.37 0.371 71.32 0.373 65.02 73.27 0.369 66.02 75.22 0.370 67.02 77.22 0.370 68.02 79.32 0.371 69.02 81.27 0.374 70.02 83.27 0.377 71.02 85.22 0.387 72.02 87.27 0.402 73.02 89.32 0.434 74.02 91.32 0.479 75.02 93.27 0.494 76.02 95.37 0.495 77.02 78.02 79.02 80.02 81.02 82.02 83.02 84.02 85.02 86.02 87.02 88.02 89.02 90.02 91.02 92.02 93.02 -9.57E-05 -5.16E-05 2.00E-04 5.74E-04 2.45E-04 -3.67E-04 - 1.19E-04 3.94E-04 2.60E-04 -1.07E-05 5.21E-OS 1.58E-04 4.30E-04 1.00E-03 7.76E-05 -1.66E-03 -1.03E-03 3.71E-04 3.87E-04 1.11E-04 1.48E-04 1.38E-04 7.31E-04 1.63E-03 1.73E-03 1.38E-03 2.71E-03 4.41E-03 5.95E-03 7.03E-03 1.04E-02 1.46E-02 1.78E-02 2.25E-02 1.78E-02 9.79E-03 5.00E-03

Answer 1

• Purity of DNA preparations can be  measured by taking  absorbance from 230nm to 320nm to detect other possible contaminants.
• However with Absorbance data given here , one can get the idea of DNA purity by applying the most common purity calculation i.e by calculating  ratio of the absorbance at 260nm divided by the reading at 280nm.

Here A260  = 0.524

  A280 = 0.283

DNA purity =  A260/A280

= 0.524/0.283

= 1.851

Good-quality DNA will have ratio  of 1.7–2.0.

In this experiment  it is coming out 1.85 thus Purity of DNA is good.

Part (ii)

Absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA.

Thus ratio of absorbance at 260nm to 230nm gives idea of  the level of salt carryover in the purified DNA.

As absorbance at A234=0.350

In the present experiment A260/A230 = 0.524/0.350 = 1.49

As a guideline, the A260/A230 is best if greater than 1.5 which is slightly lower 1.49 ,

lower ratio, indiate the salt carryover in the purified DNA than  required.