A few years ago we amplified a section of the rbcL gene by PCR, just as we did l

Question

A few years ago we amplified a section of the rbcL gene by PCR, just as we did last month. Below is a photo of the gel from one of those labs: We did the experiment the same way as this year: 1 mixed up a single Master Mix for all of the students to use along with their own plant DNA. The last three samples (not including the DNA ladders in the first and last lanes, which serve as size standards) are mine; I ran my reactions over the weekend following the lab, using a different batch of Master Mix. Now, clearly this wasn't a very successful experiment. Here's a list of the reasons we came up with for why the gel might have failed to show any PCR products from their reactions: poor preparation of DNA sample (skipped a step, wrong incubation temp or time, etc.) the Master Mix was bad (dead enzyme, forgot to add one or both primers, etc.) target DNA wasn't added (e.g. because of pipetting errors) ran the incorrect PCR protocol in the thermocycler the sample wasn't loaded into the gel the wrong percentage gel was used the gel was run too long, fragments ran off the bottom the gel was run backwards, losing the samples the DNA stain was defunct, and so samples were there but not visible Which of the above reasons seem most likely as explanations for the case of the previous year's gel? For each possibility above, explain why it seems likely (or not). Feel free to group them as appropriate: "Reasons x, y, and z couldn't be the cause because blah, blah, blah."

Explanation / Answer

a poor preparation of DNA sample (not likely) :

The subject has prepared a common master mix

b.the master mix was bad (not likely)

master mix contains all components except the template. We can see that some lanes are showing non-specific bands and most important indicator is positive control (in last three lanes).

c.target DNA was not added (not likely)

the reason is if there is no target DNA then primer dimer will be visible at bottom of lane showing no amplicon.

d. ran the incorrect pcr protocol in the thermocycler (most likely)

The most critical temperatures are those for annealing, extension and denaturation. An annealing temperature that is too low results in non-specific amplifications and primers may bind nonspecifically to the template. Too high results in no products or a low yield of product.Denaturation temperatures that are too low result in lower yields or non-specific products. If they are too high for extended periods of time they can fry the enzyme and reduce or eliminate the yield. If the extension time is too short, there will be insufficient time for complete replication of the target.Excessive extension time can allow nonspecific amplification and smearing . The likelihood of these kinds of problem is observed in our gel picture also.

moreover, if the template and primer are mismatched, particularly at the 3`-end of the primer, amplification will be reduced or eliminated. Among more closely related taxa sometimes introns are inserted within the priming site. Introns or large inserts can also be inserted between priming sites making the region too large to amplify efficiently. If the target DNA is present in relatively few copies there may be other sites competing for your primers (competitive PCR), and this may in turn significantly lower the yield. Alternatively, if the templates have a very high CG content they may not denature completely and then will not amplify well. The result is you get a no band or weak and/or fuzzy product that is much larger than the expected product and multiple fragments are amplified.

e.the sample was not loaded in the gel (likely)

no presence of primer dimer in lane showing no amplicon.

f.the wrong percentage gel was used (not likely)

we have molecular weight ladder and positive control lane that contains amplicon of respective gene.

g. the gel was run too long, the fragments ran off the bottom (not likely)

   we have positive control lane that contains amplicon of respective size.

h.the gel was run backwards, losing the samples (not likely)

The movement of band in positive lanes shows smear lagging behind the amplicon and the molecular weight marker is well separated.

i. the DNA was defunct and so samples were there but not visible (likely)

DNA extracts are often poorly extracted, quantified, degrade over time, and contain inhibitors to PCR. If the template concentration is too high, the polymerase can be inhibited due to carryover of inhibitors or inefficient denaturation. So, it is possible that the lanes containing no band might have received a sample which was not properly analysed or prepared. The result is that there may some PCR reaction which is not visible on gel due to limitation of dye used for visualizing amplicons within gels.

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