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1. (3 pts) Discuss the results of your PCR. Provide a photograph of your gel, clearly labeled. What did you expect? What did you observe? How did you interpret these results?
Obviously the answer to this question varied by group. Criteria for full credit included clear labels for each lane of the gel (including what was loaded, and what volume) and interpretation of the controls as well as the experimental result (particularly the lack of a band in the negative PCR control).
3. (2 pts) Use your gel to determine the size of your PCR product. To do this, you may either construct a standard curve as you did in week 1, or you may use the analysis software packaged with the GelDoc system. (I can consult with you about how to do the latter.) How does this size compare with the size you expected? Given the degree of accuracy that you found for this method in week 1, do you think that your PCR product is correct?
Full-credit student answers included a discussion of the estimated size of your PCR fragment vs. its expected size, and compared this to the range of accuracy that you found for gel electrophoresis in week 1.
NOTE: using the computer to do your analysis does not change the inherent precision (or lack thereof) of gel electrophoresis. The computer can measure the distance migrated by each band more precisely than you can, but the correlation between this measurement and the log of the size of the DNA fragment remains imperfect.
4. (1 pt) What was your final yield of purified PCR product, as calculated from your OD260 reading? Show your calculations.
Final yield measurements should be given in terms of total mass, not concentration. Concentration tells you how much DNA you have per unit volume, but not how much total DNA you have.
Total volume was the volume of your column eluate, 50 µl.
5. (1 pt) What was your OD260/OD280 ratio? What does this tell you about the purity of your final product?
Most of these numbers were way off from the expected value of 1.8 - 2.0. I'm not sure of the reason for this. If this were a DNA prep from cells, a value lower than 1.8 would suggest contamination with protein and a value higher than 2.0 would suggest contamination with RNA. Under these circumstances my guess is that either the spec readings were not accurate or that you had contamination with some chemical element of the kit that affected absorbance at 260 and/or 280 nm.
6. (2 pts) An alternate method for quantifying DNA is to use the intensity of ethidium staining in an agarose gel. The 1 kb DNA ladder that you used as a standard gives you a comparison. This DNA prep is 0.1 mg/ml, and about 1/10 of the mass of the DNA is comprised of the 1.6 kb fragment. How does the intensity of staining of your PCR product compare to this band? (You can "eyeball" this if you like, or you can use the analysis software to estimate it.) Using this estimate, what would you have predicted for your total QIAquick yield, assuming 100% efficiency? (Remember that you loaded 5 µl of the DNA ladder, and that you purified 7.5 times as much PCR DNA as you loaded on the gel.)
There are 80 ng of the 1.6 kb fragment in 8 µl of DNA ladder. (and 40 ng in 4 µl, and 20 ng in 2 µl). If you were using the computer, you used these values to make a standard curve and then estimate the mass of your DNA fragment from that standard curve. If you were "eyeballing" it, you compared the intensity of your PCR product band to one or more of these fragments and used that to estimate the mass of your PCR product band.
The theoretical yield from the QIAquick column is 7.5 times the mass of your PCR band in the gel, because you loaded 10 µl of PCR product in the gel and then purifed 75 µl of PCR product via the column.
7. (1 pt) Given your answers to #4 and #6, comment on the efficiency of the QIAquick procedure. If it was not very efficient, hypothesize a likely reason. (Assume that the wash buffers were made correctly this time.)
The answer to this question varied from
group to group. For the most part, the spec results suggest that
column purification was at least reasonably efficient, and that you
had a significant amount of PCR product in your eluate.
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Created by:
bkbaxter@lclark.edu
Updated: 17 Oct 00