MadSci Network: Molecular Biology
Query:

Re: Re: What can be interpreted of bands in electrophoresis from user extracted DNA

Date: Wed Jan 4 12:22:31 2006
Posted By: Sue Baker,
Area of science: Molecular Biology
ID: 1136219735.Mb
Message:

This sounds like it’s going to be a very visual experiment.  Using DNA from
onion, peas, lamb and chicken liver should yield some very interesting
banding patterns when all of your samples are digested with the same
restriction enzyme.  Restriction enzymes, also known as endonucleases, are
enzymes that recognize very specific “target sequences” in DNA and then
lyse, or cut, the DNA at that site.  For example, the endonuclease EcoRI,
derived from E. coli, recognizes the sequence GAATTC and will cleave the
DNA after the 5’guanine on each strand, in the following manner:
  
			   5’--   G/AATTC   --3’
			   3’--	C TTAA/G   --5’

Every time the enzyme encounters this sequence, the DNA is cut apart.  The
number and size of the DNA fragments will depend upon how many times this
sequence occurs within the genome and how many basepairs are in between
each sequence.  Make sense?  

Since your experiment focuses on the differences in the DNA of four
different species of both plants and animals, your choice of enzyme is not
critical, so I would suggest using EcoRI, since it is a very common enzyme
and is not too expensive, but for a list of other enzymes and their target
sequences, you can click
here. 
The buffer you will use will come with the enzyme when you order it, but be
aware the buffer comes as a 10X stock solution, so you will need to dilute
it to 1X before you use it.

Your question about how much DNA to load on a gel is a critical one and
shows you are thinking!  Oftentimes students think that more is better, but
you should remember the Goldilocks standard:  Too much DNA and your gel
will look like one big smear.  Too little DNA and your bands may be too
faint to see.  You need to load your gel JUST RIGHT.  In general, if you’re
using a miniprep kit to extract your DNA, you should use about 4µL of
extracted DNA, 1µL of EcoRI and 15µL of 1X buffer in your digestion
reaction.  After the incubation, just add 2µL of loading dye and load 20µL
onto your gel. 
Methodbook.net has a
great detailed explanation of this and a standard protocol.  You should
also bookmark
Protocol-online.org.  This is a
great resource to search for any type of protocol you might need now and in
the future.

Your final question is one that YOU need to think about:  “. . . what can
be determined from the differences seen in the bands?”  Agarose gel
electrophoresis separates the DNA fragments according to size, with the
smallest fragments traveling furthest through the gel.  From what you know
about how DNA differs (or is similar) between species, what would you
expect to see?  Do you think the plants will have similar bands?  Do you
think there will be more similarities between lamb and chicken than there
will be between lamb and peas?  What about lamb and onion?  Or do you think
that all four phenotypes (physical characteristics) are so different that
you expect no similarities between their genotypes (genetic makeup)?  I can
think of several good hypotheses you can formulate about what similarities
and differences you expect to see.  After you run your gel, compare each
pattern to see if they support your hypotheses.  Have fun!

Sue Baker, Mad Scientist
P.S.  If you get really good results, try this:  Prepare another digestion
of all of the samples again, but this time have your teacher prepare an
extra tube of one of the samples at random.  This should be labelled
“unknown.”  (Only your teacher knows its true identity.)  Then run all five
samples side-by-side on a gel and see if you can identify the mystery DNA
by comparing its banding pattern to all of the knowns.










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