Jack D. Pollard, Jr.
4/12/98
Sieving agarose is chemically modifiedto give low gelling (~35°C) and melting (~65°C) temperatures. Sievingagarose gels are poured and run like conventional agarose gels, but resolvesmall DNA fragments like nondenaturing polyacrylamide gels. They finelyresolve nucelic acid fragments less than 1000 basepairs and can distinguishfragments as small as 8 basepairs.
Materials
Sieving agarose (NuSieve GTG agarose)
TAE or TBE electrophoresis buffer,pH 8 (APPENDIX 2)
1 M NaCl
10 mg/ml tRNA or glycogen
Buffered phenol at room temperature(UNIT 2.1)
Ethanol or isopropanol
TE buffer, pH 8 (APPENDIX 2)
Additional reagents and equipmentfor agarose gel electrophoresis (CURRENT PROTOCOLSUNIT 2.5) andisolating DNA using low gelling/melting temperature agarose (UNIT 2.6of CURRENT PROTOCOLS in MOLECULAR BIOLOGY)
1. Melt 2% to 4% sieving agarosein the appropriate buffer as outlined by the manufacturer. Pour gel ofdesired size for ordinary agarose gel apparatus.
Seiving gels generally resolveDNA more slowly and with sharper resolution in TBE than in TAE. Smallerfragments (<300 basepairs) are best seperated in TBE and larger onesin TAE.
2. Load sample and run gel as foran ordinary agarose gel (CURRENT PROTOCOLSUNIT 2.5). Bromphenolblue will migrate at a rate equal to DNA fragments of <20 basepairsand xylene cyanol at a rate equal to 150 basepairs for a 4 % gel in TBEbuffer for NuSieve GTG agarose.
The gel can be run with ethidiumbromide (0.5 mg/ml) which will only cause slight decrease in DNA mobilitydue to unwinding of the helix resulting from the intercalation of the dye.
CAUTION: Ethidium bromide isa mutagen.
3. For isolation of fragment followsteps 2 to 9 of alternate protocol for low gelling/melting temperatureagarose (CURRENT PROTOCOLSUNIT 2.6).
Various manufactures have developedmethods for the removal of ethidium bromide from agarose gel slices. Spincolumns (Supelco) with a negatively charged matrix that attracts ethidiumbromide and repeals DNA offer quick, easy use along with high recovery.
COMMENTARY
Background Information
The unique pore size of sieving agarose allows separationof much smaller fragments than ordinary agarose. Sieving agarose gels inthe range of 2 to 4 % separate in the range of 8to 1000 basepairs and provide an alternative to polyacrylamide gels.Although the bands on the sieving gels are somewhat more diffuse and theresolution is slightly poorer, sieving gels are easier and faster to pourand run, making them useful for a variety of applications, such as checkingligation of linker monomers into ladders (UNIT3.16). Like low gelling/melting temperature agarose, sievingagarose is used to purify DNA fragments because of its unique propertiesof melting at 65°C and remaining liquid at ~35°C.However, DNA fragments prepared from polyacrylamide gels are generallycleaner and can be more reproducibly used as substrates for a variety ofenzymes after gel purification.
Critical Parameters
Seiving agarose does not have the integrity of regularagarose and is even more fragile than the common low gelling/melting temperatureagaroses. The manufacturers recommend that you do not use agarose concentrationsof less than 2% seiving agarose. Extreme care should be taken when pullingthe combs out of the gel, as wells tear easily. Itis usually best to remove the comb once the gel is submerged in the electrophoresistank. Because this type of agarose melts at 65°C, maintain alow voltage (6 to 7 V/cm of gel) to keep the gel from heating too much.Bromphenol blue will migrate at a rate equal to DNA fragments of <20basepairs and xylene cyanol at a rate equal to 150 basepairs
for a 4% gel in TBE buffer for NuSieveGTG agarose.
Anticipated Results
Yields are similar to conventional low gelling/meltingtemperature agarose ("70%) as long as fragments are <1000 bp. Thesegels resolve best below 500 bp.
Time Considerations
The procedure is short, especially for minigels, and canbe completed in 2 hr. After the addition of ethanol to the extracted fragment,the protocol can be interrupted for as long as desired.