Electrophoresis through agarose or polyacrylamide gels is the standard way to separate, identify and purify nucleic acid fragments. The location of the nucleic acid within the gel can be determined by using the fluorescent intercalating dye ethidium bromide. |
Agarose gels have a smaller resolving power than polyacrylamide gels but a greater range of separation - from 200 bp to >50 kb using standard gels and electrophoresis equipment. RNAs up to 10 000 kb can be separated in agarose gels using pulsed field gel electrophoresis.Polyacrylamide gels have enough resolving power to separate fragments differing by only one base pair in size, but their range is ~ 5 to 1000 bp. They are much more difficult to handle than agarose gels.
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Formaldehyde-agarose gel electrophoresis top |
Agarose gels are cast by completely melting the agarose in the desired buffer and then pouring into a mould to harden. RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode. RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form The rate of migration is determined by :- |
Molecular size of RNA |
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Agarose concentration |
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Conformation of the RNA |
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% age of agarose (w/v) in gelEfficient range of separation of linear RNA molecules - bp0.35 000 - 60 0000/61 000 - 20 0000.7800 - 10 0000.9500 - 7 0001.2400 - 6 0001.5200 - 3 0002.0100 - 2 000 |
Applied voltage. |
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Base composition of the RNA and temperature of the gel |
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Presence of intercalating dyes |
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Composition of the electrophoresis buffer |
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Apparatus for agarose gel electrophoresis |
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Polyacrylamide gel electrophoresis |
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% age acrylamide (w/v) with BIS at 1:20Effective range of separation - bpSize of RNA co- migrating with XyleneSize of RNA co- migrating withCyanolBromophenol Blue3.51 000 - 2 0004601005.080 - 500260658.060 - 4001604512.040 - 200702015.025 - 150601520.06 - 1004512 |
Polyacrylamide gels are poured between two glass plates held apart by spacers of 0.4 - 1.0 mm and sealed with tape. Most of the acrylamide solution is shielded from oxygen so that inhibition of polymerisation is confined to the very top portion of the gel. The length of the gel can vary between 10 cm and 100 cm depending on the separation required. They are always run vertically with 0.5 / 1 x TBE as a buffer. |
Three main advantages over agarose gels: |
::Resolving power is such that RNA molecules differing in length by 1 base in 500 i.e. 0.2% can be effectively separated::They can hold up to 10 mg of RNA per slot (20 mg of RNA) without loss of resolution, a much larger amount than agarose gels::The recovered RNA or RNA is extremely pure |
Two types of polyacrylamide gel in general use |
Denaturing gels : these gels are polymerised with a denaturant that suppresses base pairing in nucleic acids - this is usually urea but can be formamide. Denatured RNA migrates through the gel at a rate which is almost completely independent of its composition or sequence. These gels are used for the analysis of sequencing reactions, RNase protection assays and purification of radiolabelled RNA and RNA probes. |
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