Purpose:

TobreakuphighmolecularweighthumanplacentalDNAintofragmentsizesof500bporlesswhichcanbeusedascompetitorDNAinSouthernandinsituhybridizations.ThismethodcanalsobeusedforsonicatingsalmonspermDNA.

Timerequired:

2hoursforthesonication

Specialreagentsrequired:

• humanplacentalDNA(Sigma,Cat.#D4642)

Specialequipmentrequired:

• Asonicatorsetupwithamicro-tip.Thismethodisdesignedforusewiththesonicatoronthe8thfloorofMcDonnellScienceBuilding(GeneticsDept.).Ifyouareusinganothersonicatorpleasechecktherelevantmanuals,especiallyabouttheoutputanddutycyclelimitsonthemicro-tip.

Procedure:

1. Dissolve500mghumanplacentalDNAin100mlTEovernightatroomtemperature.

   Useasterilebeakerandkeepcovered.

2. TransferdissolvedDNAin8mlaliquotsto15mlscrew-captubesandsetupthesonicatorcontrolsasfollows:Outputcontrol="6";Dutycycle="60%";Hold="continuous".

3. Washthemicro-tipwith70%EtOHanddH2O.WipedrywithKimwipes.Placethemicro-tipimmersedhalfwayintheDNAsolution.Wearearmuffs.

4. SonicatetheDNAin1minutepulsesfor7to10minuteswitha1minutecoolingintervalbetweeneachpulse.(i.e.sonicatefor1minute,coolfor1minute).Duringthiscoolingintervalplacethetubesinanicebucket.Washthemicro-tipwith70%EtOHandddH2Obeforeandaftereachuse.

5. Combinethesamplesandrun1祃ofsonicatedDNAona0.8%TA-agarosegelat50Vfor1hourwithBRL1kbladderasaMarker.

6. ThebulkofthesonicatedDNAshouldbearound500bpinsize.Ifnot,continuesonicationandtestingsamplesonthegeluntilyouhavetherightsizefragments.

7. 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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