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Analysing and Interpreting Gel Electrophoresis Results

Agarose gel electrophoresis is used to separate and visualize DNA fragments. Interpreting gel results requires considering several factors that can affect migration patterns such as buffer composition, agarose concentration, DNA purity and concentration, voltage, and gel preparation technique. Contamination by RNA or proteins can be identified by smears above or below DNA bands respectively. Improper gel or sample preparation can prevent DNA from migrating out of wells or cause smearing. Reusing loading buffer changes its concentration and leads to smeared bands. Careful control of procedures is needed to obtain clear results.

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hely shah
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227 views

Analysing and Interpreting Gel Electrophoresis Results

Agarose gel electrophoresis is used to separate and visualize DNA fragments. Interpreting gel results requires considering several factors that can affect migration patterns such as buffer composition, agarose concentration, DNA purity and concentration, voltage, and gel preparation technique. Contamination by RNA or proteins can be identified by smears above or below DNA bands respectively. Improper gel or sample preparation can prevent DNA from migrating out of wells or cause smearing. Reusing loading buffer changes its concentration and leads to smeared bands. Careful control of procedures is needed to obtain clear results.

Uploaded by

hely shah
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Analysing and Interpreting Gel Electrophoresis

Results
Agarose gel electrophoresis is an important technique in molecular genetics since long.
The DNA bands can only be visualised using the agarose gel electrophoresis. In the genomic
research, analysing and interpreting the agarose gel electrophoresis results are very crucial.
Factor affecting the gel electrophoresis results:
 The composition and concentration of the buffer
 The concentration of the agarose gel
 The purity and concentration of the DNA
 The voltage of the electrophoresis
 Use of the buffer and agarose gel
 Preparation of the gel

Protein and RNA contamination in gel electrophoresis results


The RNA molecules are lighter than the DNA. So, the RNA migrates faster than the DNA and
DNA migrates faster than the protein because proteins are heavier than DNA. See the
Image,

Here, in the image, the smear above the DNA band indicates the contamination of
the RNA while the smear below the DNA band indicates the contamination of the protein.
Analysing and interpreting DNA gel electrophoresis results:

Image 1

See the well 9: the DNA is trying to come out from the gel but not migrated properly. Also,
the smear above the DNA is indicating the contamination of the  RNA. All other DNAs are
degraded.

Image 2

From 64 to 79, in each well DNA is trying to come out of the well but some DNA remained
inside the well. A couple of reasons are responsible for that Firstly, the wells are broken
during sample loading (see 72, 74, 75, 76, 77, 78) and secondly, the air bubbles  were
formed during the gel casting.
One another possibility in this gel is that the comb is not placed properly or the gel is
disturbed during the removal of the comb. Due to these reasons, the DNA is unable to
come out from the well.
Furthermore, smearing above the DNA indicates the high contamination of RNA into the
sample and smearing behind the DNA band (in the wells 75, 76, 77, 78, 79) indicates the
contamination of protein.
Conclusively, the DNA is not extracted properly and the gel preparation is poor.

Image 3

The DNA in the wells 50, 51, 52, 53, 54 and 55 are degraded. The concentration of the DNA
is very high in the well 59 hence it cannot come out of the well. The comb is not removed
properly from the wells 56, 59, 60, 61 and 62. The DNA samples are highly contaminated
with proteins as well as RNAs (59 to 62).

Image 4

The DNA in the wells 31, 32, 33, 34 and 35 are highly contaminated with RNA. Now see the
wells 37, 38, 39 and 40, the gel is not poured properly so the air bubbles have remained
inside the wells which hindered DNA from migrating towards the positive pole. Samples 45
and 47 are not extracted well, highly contaminated with protein and RNA.

Image 5

Now, this case is a bit different from other gels. Here the gel loading buffer is reused so
many times, therefore, the actual concentration of the buffer is changed during the
electrophoresis of this gel. Due to this reason, the buffer hindered in the migration of DNA
and smear of DNA band appeared. Also, the gel is slightly brighter than other gels because
of the fragments of other DNA (in each run some amount of DNA remains in the buffer
which appears into the next run when we re-use it).

For achieving this type of good results you have to keep in mind of several points,

 Do not re-use the gel. If necessary use only twice.


 Do not reuse the buffer. If necessary use only twice or thrice.
 Prepare buffer freshly every time for the gel as well as the electrophoresis tank.
 Preserve DNA and DNA ladders properly in the cold chain.
 Use template DNA ~30ng to 50 ng not more than that, in the PCR reaction.
 Use only 10pMol primers. Do not use the PCR reagents more than given into the
protocol.
 Use high-quality chemicals.

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