DataONE:Protocols/FindGEOreuses资讯
来自 : 蚂蚁淘
Purpose:
TobreakuphighmolecularweighthumanplacentalDNAintofragmentsizesof500bporlesswhichcanbeusedascompetitorDNAinSouthernandinsituhybridizations.ThismethodcanalsobeusedforsonicatingsalmonspermDNA.
Timerequired:
Specialreagentsrequired:
- humanplacentalDNA(Sigma,Cat.#D4642)
Specialequipmentrequired:
- Asonicatorsetupwithamicro-tip.Thismethodisdesignedforusewiththesonicatoronthe8thfloorofMcDonnellScienceBuilding(GeneticsDept.).Ifyouareusinganothersonicatorpleasechecktherelevantmanuals,especiallyabouttheoutputanddutycyclelimitsonthemicro-tip.
Procedure:
- Dissolve500mghumanplacentalDNAin100mlTEovernightatroomtemperature.
Useasterilebeakerandkeepcovered.
- TransferdissolvedDNAin8mlaliquotsto15mlscrew-captubesandsetupthesonicatorcontrolsasfollows:Outputcontrol="6";Dutycycle="60%";Hold="continuous".
- Washthemicro-tipwith70%EtOHanddH2O.WipedrywithKimwipes.Placethemicro-tipimmersedhalfwayintheDNAsolution.Wearearmuffs.
- SonicatetheDNAin1minutepulsesfor7to10minuteswitha1minutecoolingintervalbetweeneachpulse.(i.e.sonicatefor1minute,coolfor1minute).Duringthiscoolingintervalplacethetubesinanicebucket.Washthemicro-tipwith70%EtOHandddH2Obeforeandaftereachuse.
- Combinethesamplesandrun1祃ofsonicatedDNAona0.8%TA-agarosegelat50Vfor1hourwithBRL1kbladderasaMarker.
- ThebulkofthesonicatedDNAshouldbearound500bpinsize.Ifnot,continuesonicationandtestingsamplesonthegeluntilyouhavetherightsizefragments.
- TakeaspectrophotometerreADIngofthesonicatedDNAandadjusttheconcentrationto2.5mg/ml(seesamplecalculationbelow).TakeasmallaliquotoftheDNAanddilutetoproduceanOD260readingtobetween0.1and0.3.
Example:
TheOD260ofasampleis0.28at1:300dilution.Then:OD260ofundilutedsolution=0.28x300=84.0
SinceanOD260readingof1.0=50礸/mlofDNA,theconcentrationoftheundilutedsolutionis50x84=4200礸/ml,or4.2mg/ml.
Ifthepresentvolumeis100ml(420mg),addenoughTEtoadjustthefinalconcentrationto2.5mg/ml.(Inthiscase,add68mlTEtobringthefinalvolumeto168ml.)
- AdjusttheDNAconcentrationto2.5mg/mlwithTE.Storein5mlaliquotsat-20degreesC.
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