Restriction Enzyme Analysis

Restriction Enzyme Analysis

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Restriction Enzyme Analysis


Research question: Using only the information from these experiments
calculate the number and size of the fragments that would be made
using BAMH1 restriction enzyme, and calculate the migration distances
of the various fragments.

Restriction enzyme used

Base pairing fragments

Distance Travelled (cm)*

Log of base pairs*

Example

My Group

ECO R1 (6 fragments)

21226

7421

5804

5643

4878

3530

1.4

2.0

2.3

2.5

2.9

-

1.7

2.0

2.2

2.3

-

-

4.3

3.9

3.8

3.8

3.7

3.5

HIND III (7 fragments)

23130

9416

6682

4361

2322

2027

564

1.4

1.8

2.2

2.7

3.5

3.7

5.8

1.6

1.8

2.1

2.3

2.9

3.1

-

4.4

4.0

3.8

3.6

3.4

3.3

2.8

Predicted figures from my graph

BAM H1 (6 fragments)

16842

7233

6770

6527

5626

5504

1.3

2.2

2.5

2.5

2.5

2.8

4.2

3.9

3.8

3.8

3.8

3.7

Results

*All figures in table are to 2s.f.

I chose to calculate the log of the base pairs because the numbers
vary significantly, making it difficult to plot on a graph. By doing
this you can put the values into perspective for easy comparison. I
will only use the example on my graph, as my results are unclear and
inaccurate. I measured the distance from the edge of the gel, where
the well ended to the centre of each fragment.

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My Gel Ideal Gel
=================



[IMAGE][IMAGE]
==============



Conclusion
==========

From my graph I can see that the greater the distance travelled by the
fragment the fewer number of base pairs within that fragment, which is
what I would have expected. There are a few anomalies in my results in
both the EcoR1 and the Hind III; this would therefore make my final
conclusion for BamH1 slightly unreliable also. The different size
fragments in the various samples show that the base sequence in each
are completely different from each other; as the enzyme has cut at
different intervals, this also accounts for the fact that there are
different numbers of fragments in each.

Although our results were not particularly clear, from our
electrophoresis jelly we can determine that Eco R1 was the mystery
restriction enzyme in this practical.


Evaluation
----------

By drawing a line of best fit on my graph between the two known
enzymes enables me to have a rough estimation of what the Bam1 would
create. There are however, some anomalies in the results of both Eco
R1 and Hind III, even in the ideal gels. This subjects my results to
some degree of inaccuracy. These results are far better than I
obtained so to get a more accurate answer I plotted the ideal gel on
my graph.

The main problem with the gel I used, was that the smaller fragments
did not show up, this maybe because the enzyme was not entirely
successful or the fragment travelled further than the end of the gel.
The results of my gel were unclear, with little segregation between
the fragments, this meant that if I were to use this it would have
been very unreliable. I thought that including my gel to compare with
the ideal but this was also going to make it unreliable, as the
different gels have a different magnification. This can be seen in the
examples I have included. Another possible source of error in my
practical is the accuracy of my ruler, to overcome this you could use
a more accurate measuring device.

To find out whether these are accurate and reliable results you could
repeat the experiment and compare these results. Another possible
procedure would be to take the class average of each value this would
most probably give a better result.
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