Name Matt Lewis

Institution Department of Pathology University of Liverpool "garose gel ele trophoresis (#asi metho$) %a !groun$

e-mail (optional) m.lewis@liv.a .u!

"garose gel ele trophoresis is the easiest an$ ommonest way of separating an$ analy&ing DN". 'he purpose of the gel might #e to loo! at the DN"( to )uantify it or to isolate a parti ular #an$. 'he DN" is visualise$ in the gel #y a$$ition of ethi$ium #romi$e. 'his #in$s strongly to DN" #y inter alating #etween the #ases an$ is fluores ent meaning that it a#sor#s invisi#le U* light an$ transmits the energy as visi#le orange light. +hat per entage gel, Most agarose gels are ma$e #etween -../ an$ 0/. " -../ gel will show goo$ separation (resolution) of large DN" fragments (123-!#) an$ a 0/ gel will show goo$ resolution for small fragments (-.02 3!#). 4ome people go as high as 5/ for separating very tiny fragments #ut a verti al polya rylami$e gel is more appropriate in this ase. Low per entage gels are very wea! an$ may #rea! when you try to lift them. 6igh per entage gels are often #rittle an$ $o not set evenly. I usually ma!e 3/ gels. +hi h gel tan!, 4mall 783- m gels (minigels) are very popular an$ give goo$ photographs. Larger gels are use$ for appli ations su h as 4outhern an$ Northern #lotting. 'he volume of agarose re)uire$ for a minigel is aroun$ 5-21-mL( for a larger gel it may #e 01-mL. 'his metho$ assumes you are ma!ing a mini-gel. 6ow mu h DN" shoul$ I loa$, 'he #ig )uestion. 9ou may #e preparing an analyti al gel to :ust loo! at your DN". "lternatively( you may #e preparing a preparative gel to separate a DN" fragment #efore utting it out of the gel for further treatment. ;ither way you want to #e a#le to see the DN" #an$s un$er U* light in an ethi$ium#romi$e-staine$ gel. 'ypi ally( a #an$ is easily visi#le if it ontains a#out 0-ng of DN". Now onsi$er an e8ample. 4uppose you are $igesting a plasmi$ that omprises 5!# of ve tor an$ 0!# of insert. 9ou are using ; o<I (a ommon restri tion en&yme) an$ you e8pe t to see three #an$s= the linearise$ ve tor (5!#)( the 1> en$ of the insert (-.1!#) an$ the 5> en$ of the insert (3.1!#). In or$er to see the smallest #an$ (-.1!#) you want it to ontain at least 0-ng of DN". 'he smallest #an$ is 3?3-th the si&e of the un ut plasmi$. 'herefore you nee$ to ut 3-80-ng( that is 0--ng of DN" (-.0@g). 'hen your three #an$s will ontain 30-ng( 0-ng an$ A-ng of DN" respe tively. "ll three #an$s will #e learly visi#le on the gel an$ the #iggest #an$ will #e si8 times #righter than the smallest #an$. Now imagine utting the same plasmi$ with %am6I (another popular restri tion en&yme) an$ that %am6I only uts the plasmi$ on e( to linearise it. If you $igest 0--ng of DN" in this ase then the #an$ will ontain 0--ng of DN" an$ will #e very #right an$ will pro#a#ly #e overloa$e$. Too much DNA loa$e$ onto a gel is a #a$ thing. 'he #an$ appears to run fast (implying that it is smaller than it really is) an$ in e8treme ases an mess up the ele tri al fiel$ for the other #an$s( ma!ing them appear the wrong si&e also. Too little DNA is only a pro#lem in that you will not #e a#le to see the smallest #an$s #e ause they are too faint. 6aving sai$ all that( I usually $igest an$ loa$ 02B@L of the 1-@L o#taine$ from a !it miniprep. %ut you see how it $epen$s on the num#er an$ si&e of the #an$s e8pe te$. Cor PD< rea tions( it $epen$s on the PD< #ut in routine appli ations 3-20-@L shoul$ #e plenty to see the pro$u t on the gel.

+hi h om#, 'his $epen$s on the volume of DN" you are loa$ing an$ the num#er of samples. Dom#s with many tiny teeth may hol$ 3-@L. 'his is no goo$ if you want to loa$ 0-@L of restri tion $igest plus 1@L of loa$ing #uffer. +hen $e i$ing whether a om# has enough teeth( remem#er that you nee$ to loa$ at least one mar!er lane( prefera#ly two. Ma!ing the gel (for a 3/ gel( 1-mL volume) +eigh out -.1g of agarose into a 01-mL oni al flas!. "$$ 1-mL of -.18'%; ( swirl to mi8. It is goo$ to use a large ontainer( as long as it fits in the mi rowave( #e ause the agarose #oils over easily. Mi rowave for a#out 3 minute to $issolve the agarose. 'he agarose solution an #oil over very easily so !eep he !ing it. It is goo$ to stop it after B1 se on$s an$ give it a swirl. It an #e ome superheate$ an$ NE' #oil until you ta!e it out whereupon it #oils out all over you han$s. 4o wear gloves an$ hol$ it at arms length. 9ou an use a #unsen #urner instea$ of a mi rowave - :ust remem#er to !eep wat hing it. Leave it to ool on the #en h for 1 minutes $own to a#out A-FD (:ust too hot to !eep hol$ing in #are han$s). If you ha$ to #oil it for a long time to $issolve the agarose then you may have lost some water to watervapour. 9ou an weigh the flas! #efore an$ after heating an$ a$$ in a little $istille$ water to ma!e up this lost volume. +hile the agarose is ooling( prepare the gel tan! rea$y( on a level surfa e. "$$ 3@L of ethi$ium #romi$e (3-mg?mL) an$ swirl to mi8 'he reason for allowing the agarose to ool a little #efore this step is to minimise pro$u tion of ethi$ium #romi$e vapour. ;thi$ium %romi$e is mutagenic an$ shoul$ #e han$le$ with e8treme aution. Dispose of the ontaminate$ tip into a $e$i ate$ ethi$ium #romi$e waste ontainer. 3-mg?mL ethi$ium #romi$e solution is ma$e up using ta#lets (to avoi$ weighing out pow$er) an$ is store$ at BFD in the $ar! with 'EGID la#els on it. Pour the gel slowly into the tan!. Push any #u##les away to the si$e using a $isposa#le tip. Insert the om# an$ $ou#le he ! that it is orre tly positione$. 'he #enefit of pouring slowly is that most #u##les stay up in the flas!. <inse out the flas! imme$iately. Leave to set for at least 5- minutes( prefera#ly 3 hour( with the li$ on if possi#le. 'he gel may loo! set mu h sooner #ut running DN" into a gel too soon an give terri#le-loo!ing results with smeary $iffuse #an$s. Pour -.18 '%; #uffer into the gel tan! to su#merge the gel to 021mm $epth. 'his is the running #uffer. 9ou must use the same #uffer at this stage as you use$ to ma!e the gel. ie. If you use$ -.A8'%; in the gel then use -.A8'%; for the running #uffer. <emem#er to remove the metal gel-formers if your gel tan! uses them. Preparing the samples 'ransfer an appropriate amount of ea h sample to a fresh mi rofuge tu#e. It may #e 3-@L of a 1-@L PD< rea tion or 1@L of a 0-@L restri tion en&yme $igestion. If you are loa$ing the entire 0-@L of a 0-@L PD< rea tion or en&yme $igestion (as I often $o) then there is no

nee$ to use fresh tu#es( :ust a$$ in the loa$ing #uffer into the PD< tu#es. +rite in your la#-#oo! the physi al or$er of the tu#es so you an i$entify the lanes on the gel photograph. "$$ an appropriate amount of loa$ing #uffer into ea h tu#e an$ leave the tip in the tu#e. "$$ -.0 volumes of loa$ing #uffer( eg. 0@L into a 3-@L sample. 'he tip will #e use$ again to loa$ the gel. Loa$ the first well with mar!er. I store my mar!ers rea$y-mi8e$ with loa$ing #uffer at BFD. I !now to loa$ 0@L an$ how mu h DN" is in ea h #an$. 4ee #elow for more on this. "voi$ using the en$ wells if possi#le. Cor e8ample( If you have 30 samples an$ 0 mar!ers then you will use 3B lanes in total. If your om# forme$ 37 wells then you will not #e using B wells. It is #est to not use the outer wells #e ause they are the most li!ely to run a#errantly. Dontinue loa$ing the samples an$ finish of with a final lane of mar!er I loa$ gels from right to left with the wells fa ing me. 'his is #e ause gels are pu#lishe$( #y onvention( as if the wells were at the top an$ the DN" ha$ run $own the page. If this seems onfusing then you an loa$ left to right with the wells fa ing away from you. Dlose the gel tan!( swit h on the power-sour e an$ run the gel at 1*? m. Cor e8ample( if the ele tro$es are 3- m apart then run the gel at 1-*. It is fine to run the gel slower than this #ut $o not run any faster. "#ove 1*? m the agarose may heat up an$ #egin to melt with $isastrous effe ts on your gel>s resolution. 4ome people run the gel slowly at first (eg. 0*? m for 3minutes) to allow the DN" to move into the gel slowly an$ evenly( an$ then spee$ up the gel later. 'his may give #etter resolution. It is EH to run gels overnight at very low voltages( eg. -.012-.1*? m( if you want to go home at 33 E> lo ! alrea$y. Dhe ! that a urrent is flowing 9ou an he ! this on the power-sour e( the milliamps shoul$ #e in the same #all-par! as the voltage( #ut the the #est way is to loo! at the ele tro$es an$ he ! that they are evolving gas (ie. #u##les). If not then he ! the onne tions( that the power-sour e is plugge$ in et .et . 'his has #een !nown to happen if people use water instea$ of running #uffer. Monitor the progress of the gel #y referen e to the mar!er $ye. 4top the gel when the #romophenol #lue has run 5?B the length of the gel. 4wit h off an$ unplug the gel tan! an$ arry the gel (in its hol$er if possi#le) to the $ar!-room to loo! at on the U* light-#o8. 4ome gel hol$ers are not U* transparent so you have to arefully pla e the gel onto the glass surfa e of the light-#o8. U* is carcinogenic an$ must not #e allowe$ to shine on na!e$ s!in or eyes. 4o wear fa e prote tion( gloves an$ long sleeves. Loa$ing #uffers 'he loa$ing #uffer gives olour an$ $ensity to the sample to ma!e it easy to loa$ into the wells. "lso( the $yes are negatively harge$ in neutral #uffers an$ thus move in the same $ire tion as the DN" $uring ele trophoresis. 'his allows you to monitor the progress of the gel. 'he most ommon $yes are #romophenol #lue (4igma %7-0A) an$ 8ylene yanol (4igma GB30A). Density is provi$e$ #y gly erol or su rose.

Typical recipe

01mg #romophenol #lue or 8ylene yanol Bg su rose 60E to 3-mL

'he e8a t amount of $ye is not important 4tore at BFD to avoi$ moul$ growing in the su rose. 3-mL of loa$ing #uffer will last for years. %romophenol #lue migrates at a rate e)uivalent to 0--2B--#p DN". If you want to see fragments anywhere near this si&e (ie. anything smaller than A--#p) then use the other $ye #e ause the #romophenol #lue will o#s ure the visi#ility of the small fragments. Gylene yanol migrates at appro8imately B!# e)uivalen e. 4o $o not use this if you want to visualise fragments of B!#. 4i&e mar!ers 'here are lots of $ifferent !in$s of DN" si&e mar!ers. In the ol$ $ays the heapest $efine$ DN" was from #a teriophage so alot of mar!ers are phage DN" ut with restri tion en&ymes. Many of these are still very popular eg( lam#$a 6in$III( lam#$a PstI( PhiG3.B 6aeIII. 'hese give #an$s with !nown si&es #ut the si&es are ar#itrary. Dhoose a mar!er with goo$ resolution for the fragment si&e you e8pe t to see in you sample lanes. Cor e8ample( for tiny PD< pro$u ts you might hoose PhiG3.B 6aeIII #ut for A!# fragments you woul$ hoose lam#$a 6in$III. More re ently( ompanies have starte$ pro$u ing la$$er mar!ers with #an$s at $efine$ intervals( eg. -.1( 3( 3.1( 0( 0.1!# an$ so on up to 3-!#. If you !now the total amount of DN" loa$e$ into a mar!er lane( an$ you !now the si&es of all the #an$s( you an al ulate the amount of DN" in ea h #an$ visi#le on the gel. 'his an #e very useful for )uantifying the amount of DN" in your sample #an$s #y omparison with the mar!er #an$s. It is goo$ to loa$ two mar!ers lanes( flan!ing the samples. Lots of ompanies sell DN" si&e mar!ers. It pays to shop aroun$ for the heapest. Eften the lo al !it hen-sin! #iote h ompany sells e8 ellent mar!ers. . '%; '%; stan$s for 'ris %orate ;D'". People also use '"; ('ris " etate ;D'"). Ma!e up a 3-8 sto ! using heap reagents. Do not use e8pensive >analyti al gra$e> reageants. Dheap 'ris #ase an$ #ori a i$ an #e #ought in #ul!. <e ipe for 0L of 3-8'%;

037g 'ris #ase 33-g %ori a i$ I.5g ;D'"

Dissolve the ingre$ients in 3.IL of $istille$ water. p6 to a#out 7.5 using NaE6 an$ ma!e up to 0L. . No responsi#ility is assume$ #y metho$#oo!.net for any in:ury an$?or $amage to persons or property as a matter of pro$u ts lia#ility( negligen e or otherwise( or from any use or operation of any metho$s( pro$u ts( instru tions or i$eas ontaine$ in the material herein. It is the users responsi#ility to ensure that all pro e$ures are arrie$ out a or$ing to appropriate 6ealth an$ 4afety re)uirements. http://www.methodbook.net/dna/agarogel.html