In June 2011, the Year 13 Biology
classes amplified chloroplast DNA they had extracted from
plants.
This process was done using the polymerase chain reaction (PCR).
The DNA was copied from the chloroplasts in the leaves (cpDNA). The two primers used
targeted a section of the variable cpDNA between the conserved tRNA
genes in the chloroplast.
The denaturation (94oC); annealing (55oC); extension (72oC) was achieved by moving the samples from one waterbath
to the next every 30 seconds for thirty cycles.
To understand what was happening
see this You Tube video.
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Dye was added before loading the amplified DNA into the wells in the gel. |
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The DNA was run in an agarose gel by electrophoresis. The gel works like a sieve,
with small molecules moving quickly from the wells where they started, the large
molecules dragging along behind. This separates them according to size.
A reference ladder (ruler) of known DNA sizes was run on the same gel to estimate
how big the extracted DNA fragments were. |
The amplified DNA had to be delivered to the tiny wells in the agarose gel with care, to avoid damaging the soft medium. Micropipettes containing about 10 microlitres of solution were used.
RESULTS:
These are photographs of gels made as shown in the picture above. This is how they appear after undergoing electrophoresis for a couple of hours and being stained with 0.04% Azure A.
On the right hand side of the gels the DNA ladder was loaded. The sizes of the fragments in base-pairs is given. All samples were put into the wells at the bottom.
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Year 13 Biology Students' Prep.
By only amplifying one piece of the cpDNA it is not possible to obtain much information regarding the evolutionary relationships of these plants, but the technique of DNA profiling can be learnt.
Write a conclusion to this investigation using the left hand gel to indicate:
which plant appears to be least closely related to the others
your estimation of the size of the largest and smallest chloroplast DNA fragments
the chives and onion DNA seem to be present in smaller amounts than the other two, what is the likely reason for this?
The right hand gel was made to compare the orchid, a recently-evolved angiosperm, with some lower plants. We had no success with the moss or the fern, but there appears to be a slight difference between the ancient liverwort bryophyte and the orchid. What does this indicate that supports the orchid being a more highly-evolved plant?
Our thanks to Cottage Flowers of Broadway, Palmerston North for providing the complimentary orchid.
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