DGGEFORSymbiodinium

ByCamilaGranadosCifuentes,IMaGeSLab (ModifiedfromDr.ToddLaJeunesseand Dr.JuanArmandoSanchezBIOMMAR)

This protocol explains the stepbystep process for running a DGGE for PCR productsoftheSymbiodiniumITS2.DGGE,denaturinggradientgelelectrophoresis, is a technique that uses electrophoresis and denaturing agents, such as urea and formamide, to denature DNA. Thus, the denaturing agents are in a gradient throughoutthegelwhiletemperatureandvoltagearekeptconstant.DGGEenables the researcher to separate PCR products based on sequences, as opposed to size when doing agarose or polyacrylamide gel electrophoresis. In the case of Symbiodinium,DGGEhasbeenusedtodifferentiatebetweenthedifferentstrainsof Symbiodinium(e.g.LaJeunesse,2001;LaJeunesse,2002;LaJeunesseetal.,2005).

PCR

The primers for the amplification of the ITS2 from Symbiodinium are

ITSintfor25GAATTGCAGAACTCCGTG3andITS2CLAMP5CGCCCGCCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GGG ATC CAT ATG CTT AAG TTCAGCGGGT3(LaJeunesse&Trench,2000). Amplification is obtained from a touchdown PCR. The touchdown protocol consistedofaninitialdenaturingstepat92Cfor3min,21cyclesat92C for 30s, 62 C for 40s, and 72 C for 30s, decreasing each cycle 0.5 C, followedby15cycleswitha52Cannealingstep,andafinalextensionat72 Cfor10min.Please,followthesesteps.

Theconditionsare:1Loftemplateataconcentrationbetween310ng/L,

10LofGoTaqGreenMasterMix1X(2XGreenGoTaqreactionbuffer,400 M each dNTP, 3.0 mM MgCl2, GoTaq DNA polymerase, Promega), 0.25 M forwardprimer,0.75Mreverseprimer,andaddedMilliQwaterforafinal volume of 20 L per reaction. Although you may vary the final volume and concentration of primers, you should ALWAYS keep a 1:3 ratio because the reverse primer with clamp will tend to form primer dimmers. This is why youalwaysaddanexcessofreverseprimer. Concentration of template varies depending upon quality of DNA, source of DNA (from a host, freeliving, or culture). Test different dilutions. Once you havegoodbands,youarereadytogo.

THEGEL

Day0SOLUTIONS
1. Prepareallyourstocksolutionsorbymakingsureyouhaveenoughofthem. Thesolutionsare: BufferTAE20X 55gsodiumacetate 7.5gEDTA 95gTrisbase MixwithdiH2OandadjusttopH7.4withHCL(37%).Bringtoafinalvolume of1L. 10%APS 2mlAPS 18mldiH2O 40%Acrylamide 40gAcrylamide 1.09gBisacrylamide Bringtoafinalvolumeof100ml. 8%Acrylamide 40%xVi=8%x100ml Vi=20mlacrylamide40%and80mldiH2O Formamide Original Urea Original TEMED Original

USENITRILEGLOVESATALLTIMES!

Day1AMCASTINGTHEGEL

(*Iwouldrecommendrunningamaximumof2gelsatatime) 1. Startbypreparingyour100%denaturingsolutionasfollows: Finalvolume 40%Acrylamide Formamide Urea 20XTAE diH2O 30mL(2gels) 6mL 12mL 12.62g 1.5mL 4.5mL 15mL(1gel) 3mL 6mL 6.31g 0.75mL 2.25mL 2. PreparetheglassplatesandthawAPSwhiletheureadissolves. 3. Cleanbothsurfacesoftheglasseswith100%EtOHandletitairdry. 4. Checkthattheyellowgelwrapgasketisdryandhasnoholes.Placeitaround thethinnerroundintwocornersglassplate.Makesureitistightagainstthe glass and the corners match perfect with the cuts of the yellow gel wrap gasket.Payattentiontotheraiseononesideoftheyellowgelwrapgasket. ThiswillbeyourINNERpart. 5. Placeonespaceroneachsideoftheglassplate,nexttotheraiseoftheyellow gelwrapgasket(INNERside).Theyhavetobeinastraightline. 6. Putthesquaredcornerglassplateontopofthecurvedone.Ithastomatch perfectlyinthebottomandsidestoavoidleaking. 7. Usethecastingclamps(closedclamps)tojoinbothglassplates.(*Isuggest startingfromthebottompartoftheglassplatesandthenplacetheoneson theside). 8. CheckthattherearenoleakswithMilliQH2O.Ifthereareleaks,dissemble everything,dryandstartagainuntilyouhavenoleaks. 9. WearenowgoingtopreparethegradientfortheDGGE.InthecaseofITS2of Symbiodinium we use 4580%, using 8% acrylamide and 100% acrylamide, which consisted of 7 M urea and 40%deionizedformamide(whichshould bedissolvedbynow). 10. Marked are two falcon tubes with 45 and 80 (if not, go ahead and mark them). 11. In the 45tube, add 6.3mL of 8% (0%) acrylamide and 5.2mL of 100% denaturingacrylamidesolution(theonethatyoujustdissolved).

 12. In the 80 tube, add 2.3mL of 8% (0%) acrylamide and 9.2mL of 100% acrylamide(theonethatyoujustdissolved). 13. BEFOREaddingtheTEMEDandAPS,preparethegradientmaker,thepump (if not doing by gravity) and make sure the glassassemble is empty. The silicone tubing from the gradient maker should connect to the glass assemble.Placeasmallmagnetontheleftholeofthegradientmakerandput thegradientmakeronashaker.Checkthatbothvalvesofthegradientmaker areclosed!(Theonethatgoestothetubingandtheonethatconnectsboth chambers). 14. Onceyouhaveeverythingtopouryourgel,add5LofTEMEDand95Lof APStoEACHgradientsolution,45%and80%. 15. Immediatelyplacethemixinthegradientmaker.Itisimportantthatyouput the 80% gradient on the LEFT chamber of the gradient maker. This is because you greater percentage of denaturing solution will be in the lower partofthegel. 16. Pourthegelbyopeningthevalvethatgoestothetubing.Thiswillmakethe 80%denaturingsolutiontobepouredFIRST. 17. Whenthelevelofthe80%denaturingsolutionreachesthesilverstickeron the gradient maker, open the middle valve, allowing the two denaturing solutionstomix. 18. Placethecombs.Nobubblesshouldbeseennexttothecombs.Incasethat youhavebubbles,removethecombsandplacethemagainslowly. 19. Overflow the volume of gel. This way, you will ensure that, upon polymerization,thevolumeofthegelwillbeenoughtostillhavewells. 20. When you finish pouring, immediately place the gradient maker in running watertoavoidpolymerizationinthetubing. 21. Repeatfromsteps1120forthesecondgel.

DAY1PMLOADINGANDRUNNINGTHEDGGE
22. ONEHOURBEFORELOADING,Fillthetankwith19LofdiH2Oand1Lof20X TAEforarunningbufferof1XTAE.Heatto60C. Note:toknowthetimetostartrunning,keepinmindthatitwillrunfor14 hours. * My advice will be to start early on day 2 for staining, visualization andexcisingbands.Thus,Isuggeststartrunningat6p.m.tothelatest.Inthis case,startwarmingat4:00p.m.,prerunfrom5:005:30p.m.,andloadfrom 5:306:00p.m. 23. Remove the casting clamps, the combs and the gel wrap gasket only in the lowerpartofthegel. 24. Labelthegelse.g.Gel1andputanarrow.Thiswillhelpyouthenextdayto rememberwhichgelyouloadedfirstandthedirectioninwhichyouloaded (seesteps33and39). 25. Place gel in the gel cassette and attach with the running clamps (opened clamps). If you are running only one gel, make sure you put a set of glass platesintheothersideofthegelcassette.Thiswillcreateapoolofbuffer constantlypumpbyatubing.Thepoolwillbeformedinbetweentheglass plates,whichisimportantforkeepingtheelectrodewetduringtherun. 26. Connect the cable and the hose to the gel cassette. Make sure, buffer is runningthroughthehoseandthepool,mentionedabove,isformed. 27. Prerunat100Vfor30min.CleanthewellsusingaPasteurpipettewiththe samebufferofthetank. 28. Load your samples. Clean well by well with buffer as you load. This will ensurethattheentiresamplewillgotothebottomofthewellandyouwill haveabetterrunofthesampleinthegel. 29. Runfor14h,100V,60C.

DAY2VISUALIZATIONANDBANDEXCISING
30. MakeEtBrstainingbath:1LofdiH2Oand120lofconcentratedEtBr. 31. Turnoffpowersupply,unplugeverythingfromthegelcassette,emptypool inbetweenglassplates,andpullouteverythingfromtank. 32. Removegelwrapgasketandspacers. 33. Carefully separate the glass plates. Make sure you know the DIRECTION in whichyouloaded;thisiscriticalinthisstepandinstep39. ` Note: An easy way to separate both glass plates is by using a spatula or something similar that you can slide in between the glass plates. Once you separateoneside,therestwillcomeouteasily. 34. PlaceinEtBrforstainingfor20min. 35. Label0.5mLtubesandcovereachtubewithaluminumfoil.Youmayhaveup to40bandspergel. 36. PrepareanothercontainerwithdiH2Oonly. 37. Placegelindestainingbathfor20min. 38. Inthesecond20minutewait,finishlabelingyourtubesandcoveringthem withaluminumfoil.Youmayalsowanttoprepareyourmaterialforexcising bands:tipsnexttothetransiluminator,TBE1X(towetthegel,seestep42), asmallbeakerwith30mLof100%EtOH,andtweezers. 39. Placegelintransiluminator. Note: In this step it is also critical to know the DIRECTION in which you loaded.Toremovethegelfromtheglassplate,makesurethegeliswetfrom thebath.Puttheentiregelonthetransiluminatorandmoveitalittlebit,so that water comes in between the gel and the glass plate. As with step 33, once one part of the gel detaches from the glass plate, the rest will peel off easily. 40. Takeapictureofyourgelandsaveitimmediately! 41. Label the bands in your image. This will guide you through all the excising process.

42. Before excising bands, wet your gel. This will make band excising much easierandfaster! 43. Usethetipstocutthebands.Useanewtipforeachband.Aneasywaytocut the bands is by drawing a rectangle around the band, but start by cutting the longer sides of the rectangle (up and down). Then cut the sides (left and right). Start excising you bands from the faintest ones to the brightest ones.Withthehelpofthetip,puttheexcisedbandinadifferentpartofthe gel. Use the tweezers to put the bands in the tubes. To clean the tweezers, submergethemin100%EtOH.DrywithKimwipebeforepickingthebands. Reduce,asmuchaspossible,theamountoftimeyouhavetheUVlighton.

Note:Tipsaregoodtousebecauseyoucanputasmuchpressureasyouwant withoutscratchingthetransiluminator.Doitaccuratelyandfast. 44. Repeatstep3243forasecondgel. 45. Afteryoufinishcuttingallthebands,add30LofnucleasefreeH2Otoeach band.Usingatip,squeezethebandinthewaterandmakesurethebandisin thewater. 46. Leaveshakingovernightcoveredwithfoilatamediumspeed.

DAY34PRECIPITATION
47. Transfer the liquid (NOT THE GEL, since it will inhibit the PCR) to a correspondinglabeled1.5mLtube. 48. Add800Lofcold100%moleculargradeEtOH. 49. Putat20Covernight. 55. Drycompletely(avoidoverdrying). 56. Whencompletelydry,add30LofnucleasefreeH2OorTEpH8.0. 51. DiscardtheEtOH. 52. Add300Lofcold70%EtOH. 53. Centrifugeat12,000rpmfor10minat4C. 54. DiscardtheEtOH. 50. Thenextday,centrifugeat12,000rpmfor30minat4C.

DAY4(orlater)REAMPLIFICATION
57. Reamplify the bands by adding only 1L of template. The primers used are ITSintfor2 (see PCR) and the ITSRev 5 GGGATCCATATGCTTAAGTTCAGCGGGT3(Colemanetal.,1994). 58. The conditions are: 1 L of template, 10 L of GoTaq Green Master Mix 1X (2XGreenGoTaqreactionbuffer,400MeachdNTP,3.0mMMgCl2,GoTaq DNA polymerase, Promega), 0.25 M ITSintfor2 primer, 0.25 M ITSRev primer,andaddedMilliQwaterforafinalvolumeof20Lperreaction. 59. Theprotocolconsistedof92Cfor3min,35cyclesat92Cfor30s,52Cfor 40s,and72Cfor30s,andafinalextensionat72Cfor10min.