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Electrophoresis Tutorial

Key Concepts

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Separating DNA Fragments

DNA fragments are negatively charged due to the negatively charged phosphate groups making up the backbone of the molecule so all the fragments in the sample will migrate towards the positively charged electrode.

Electrophoresis can therefore be used to separate DNA fragments based primarily on the size of the fragment.

The results of an electrophoresis experiment to separate DNA fragments are represented below:

sample loaded herefragment
X
fragment
Y
Negative Electrode



Positive Electrode

Both fragments, X and Y, are negatively charged so they have migrated towards the positive electrode.

Fragment X is expected to have the greater molecular mass since it has travelled the least distance.
Fragment Y is expected to have the lower molecular mass since it has travelled the greatest distance.

Fragment X will be larger than fragment Y.

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Separating Amino Acids

Glycine (symbol gly) is an amino acid.

The isoelectric point for glycine is at pH 6.1.

At pH 6.1 glycine has no overall charge.

H2N - H
|
C
|
H
- COOH
Glycine will remain stationary during electrophoresis.
At pH < 6.1 glycine has a positive charge.
+H3N - H
|
C
|
H
- COOH
Glycine will migrate towards the negative electrode during electrophoresis.
At pH > 6.1 glycine has a negative charge.
H2N - H
|
C
|
H
- COO-
Glycine will migrate towards the positive electrode during electrophoresis.

Cysteine (symbol cys) is also an amino acid.

The isoelectric point for cysteine is at pH 5.0.

At pH 5.0 cysteine has no overall charge.

  CH2-SH
H2N -
 
|
C - COOH
|
H
Cysteine will remain stationary during electrophoresis.
At pH < 5.0 cysteine has a positive charge.
  CH2-SH
+H3N -
 
|
C - COOH
|
H
Cysteine will migrate towards the negative electrode during electrophoresis.
At pH > 5.0 cysteine has a negative charge.
  CH2-SH
H2N -
 
|
C - COO-
|
H
Cysteine will migrate towards the positive electrode during electrophoresis.

If a mixture containing both glycine and cysteine is buffered at pH 5.5 and then undergoes electrophoresis, the results of the experiment could be respresented as:

glycine
(positive charge)
sample loaded here cysteine
(negative charge)
Negative Electrode



Positive Electrode


At pH 5.5 glycine is positively charged so it migrates towards the negative electrode.
At pH 5.5 cysteine is negatively charged so it migrates towards the positive electrode.

If a mixture containing both glycine and cysteine is buffered at pH 7.0 and then undergoes electrophoresis, the results of the experiment could be represented as:

sample loaded here cysteine
(negative charge)
glycine
(negative charge)
Negative Electrode



Positive Electrode


At pH 7.0 both glycine and cysteine carry a negative charge so both will migrate towards the positive electrode.
Glycine has a slightly lower molecular mass than cysteine and could be expected to travel a little bit further.

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