Editing Agarose gel electrophoresis (section)

==Properties of agarose gel==
[[File:Two percent Agarose Gel in Borate Buffer cast in a Gel Tray (Front, angled).jpg|right|thumb|An agarose gel cast in tray, to be used for gel electrophoresis]]

[[Agarose]] gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass.<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual |author1=Joseph Sambrook |author2=David Russell |volume=1 |edition=3rd |chapter=Chapter 5, protocol 1  |page=5.4 |isbn=978-0-87969-577-4 }}</ref> The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state. The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of {{convert|35|–|42|C|F}} and a melting temperature of {{convert|85|–|95|C|F}}. Low-melting and low-gelling agaroses made through chemical modifications are also available.

Agarose gel has large pore size and good gel strength, making it suitable as an anticonvection medium for the electrophoresis of DNA and large protein molecules. The pore size of a 1% gel has been estimated from 100&nbsp;nm to 200–500&nbsp;nm,<ref name="zimm"/><ref name=viovy>{{cite journal |journal=Reviews of Modern Physics |volume= 72 |issue= 3 |pages= 813–872 |year=2000 |title=Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms|author =Jean-Louis Viovy  |doi=10.1103/RevModPhys.72.813 |bibcode=2000RvMP...72..813V}}</ref> and its gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis.<ref name="serwer"/> Low-concentration gels (0.1–0.2%) however are fragile and therefore hard to handle. Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a greater range of separation, and is therefore used for DNA fragments of usually 50–20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is around 750 kb, but resolution of over 6 Mb is possible with [[pulsed field gel electrophoresis]] (PFGE).<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual |author1=Joseph Sambrook |author2=David Russell |volume=1 |edition=3rd |chapter=Chapter 5, protocol 1 |page=5.2–5.3  |isbn=978-0-87969-577-4 }}</ref> It can also be used to separate large proteins, and it is the preferred matrix for the gel electrophoresis of particles with effective radii larger than 5–10&nbsp;nm. A 0.9% agarose gel has pores large enough for the entry of [[bacteriophage T4]].<ref name="serwer">{{cite journal |title=Agarose gels: Properties and use for electrophoresis |author =Philip Serwer |journal=Electrophoresis |volume = 4 |issue =6 |pages =375–382 |year= 1983 |doi=10.1002/elps.1150040602|s2cid =97819634 }}</ref>

The agarose polymer contains charged groups, in particular [[pyruvate]] and [[sulfate]].<ref name="lonza">{{cite web |url=http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Appendix_B_-_Agarose_Physical_Chemistry.pdf |archive-url=https://ghostarchive.org/archive/20221009/http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Appendix_B_-_Agarose_Physical_Chemistry.pdf |archive-date=2022-10-09 |url-status=live |title=Appendix B: Agarose Physical Chemistry |work=Lonza Group }}</ref>  These negatively charged groups create a flow of water in the opposite direction to the movement of DNA in a process called [[electroendosmosis]] (EEO), and can therefore retard the movement of DNA and cause blurring of bands. Higher concentration gels would have higher electroendosmotic flow. Low EEO agarose is therefore generally preferred for use in agarose [[gel electrophoresis of nucleic acids]], but high EEO agarose may be used for other purposes. The lower sulfate content of low EEO agarose, particularly low-melting point (LMP) agarose, is also beneficial in cases where the DNA extracted from gel is to be used for further manipulation as the presence of contaminating sulfates may affect some subsequent procedures, such as [[ligation (molecular biology)|ligation]] and [[Polymerase chain reaction|PCR]].  Zero EEO agaroses however are undesirable for some applications as they may be made by adding positively charged groups and such groups can affect subsequent enzyme reactions.<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual |author1=Joseph Sambrook |author2=David Russell |volume=1 |edition=3rd |chapter=Chapter 5, protocol 1 |page=5.7 |isbn=978-0-87969-577-4 }}</ref>   Electroendosmosis is a reason agarose is used in preference to [[agar]] as the [[agaropectin]] component in agar contains a significant amount of negatively charged sulfate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix.  However, for some applications such as the electrophoresis of serum proteins, a high EEO may be desirable, and agaropectin may be added in the gel used.<ref>{{cite book |url=https://books.google.com/books?id=z-bZ_FZHqRcC&pg=PA7 |title=Protein Electrophoresis in Clinical Diagnosis|first= David |last=Keren |pages=7–8 |publisher=CRC Press |date=26 September 2003|isbn=978-0340812136 }}</ref>