StreamlinedDNAExtractionProtocol

ThismethodisderivedfromaproceduredevelopedbyTobyBradshawandthePoplarMolecularGeneticsCooperative.WehavetestedtheprocedurewithavarietyofPopulusspecies,aswellastobaccoandArABIdopsis.TheresultingDNAisofsufficientlyhighqualityforPCR(includingRAPD),restrictiondigests,andligationreactions.However,itisextemelyimportanttousetheyoungestleavesavailableforPopulus,asDNAfromolderleavesisoftencontaminatedwithcompoundsthatcaninterferewithenzymaticreactions.Expectedyieldis5-30ug,dependingonthequalityandquantityofthefoliage.Yieldscanbeincreasedbeyond30ugbyusingmultipleyoungleavesandincreasingthevolumesofthegrindingandextractionbuffers.

1. AddDiethylDithiocarbamicAcidSodiumsalt(4mg/ml)andRNAaseA(100ug/ml)toanaliquotofGrindingBuffer,andB-mercaptoethanol(1%)toanaliquotofLysisBuffer(seebelow).
2. Useasinglenewlyemerged,stillrolled-upleaf(~10mg,thenewerthebetter).Placefoliageinmicrofugetubesinaliquidnitrogenbath.
3. Addapproximately200to400ulofliquidnitrogentoatubecontainingfoliage.Grindapprox.10-15secondsusingamotorizedpestle.Stopgrindingwhenliquidnitrogenevaporatesandtissuedefrosts.
4. Add200ulGrindingBuffer.Grindanother10secondsuntiltissueiswell-homogenized.
5. Placetubeinhotwaterbath(~40-65deg.)andincubatewhilegrindingothersamples.Eachsampleshouldbeincubatedatleast10min.
6. Add200ulLysisBuffertoeachtube.Mixbyinvertingseveraltimes.
7. Aftergrindingallsamples(typicallyasmanyaswillfitinmicrofuge),incubateat65deg.for30-60min.Every10to15minutes,mixbyinvertingtubesseveraltimes.
8. Addequalvolumeofphenol:chloroform:IAA(25:24:1).Mixwellbyinvertingatleast10times.Spin8-10min.at12,000rpmatroomtemperature.
9. Remove50-70%ofsupernatanttonewtube,takingcaretoavoidinterface.Ifsupernatantiscloudyorcontaminatedwithinterface,gotostep10.Otherwiseskipto12.
10. (Optional)Addanequalvolumeofchloroform,mixwellbyinvertingatleast10times.Spin5min.at12,000rpmatroomtemperature.
11. Remove50-70%ofsupernatanttonewtube,takingcaretoavoidinterface.
12. Add2/3volumeofisopropanol.Mixbyinverting.
13. Incubate15-30min.oniceorroomtemperature.
14. Spininmicrofuge5-10minutesatroomtemperature.
15. Decantsupernatantandblottubesonkimwipe.
16. Dryinspeed-vacfor2-4minutesuntilnoisopropanolodorisapparent.
17. Add50ulTEbuffer(10:1),pH8.Letsitatroomtemperaturefor10-20min.,orat4deg.overnight(preferred).Mixbyflickingtubeseveraltimes.

100mMTrispH8

20mMEDTApH8

4mg/mldiethyldithiocarbamicacid,sodiumsalt,addedjustpriortouse.

100ug/mlRNAaseA,addedjustpriortouse

LysisBuffer

100mMTrispH8

20mMEDTApH8

1MNaCl

2%SDS

1%B-mecaptoethanol,addedjustpriortouse

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