Lab practical report on Hot shot DNA extraction

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER:Prof NJAGI/Dr OKUN/Dr RUNO.
HANDING DATE: 8 /11/2016
SUBJECT: PRACTICAL REPPORT ON HOT SHOT DNA EXTRACTION
METHOD
Elvis
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2. Objectives
1. To isolate DNA fromfishfins.
2. To obtainPCR-qualityDNA fromthe fishfins.
BACKGROUND
DNA,the buildingblockof life isthe geneticmaterial forall livingorganismsanditcontains information
that iscrucial forheredity.DNA isolationisnecessaryforgeneticanalysisincludingscientific,medical,or
forensicpurposes.Presenceof lipids,proteins,polysaccharidesandsome otherimpuritiesinthe DNA
preparationcaninterfere withDNA analysismethodsbyreducingthe qualityof DNA, whichleadstoits
shorterstorage life.DNA canbe isolatedfromanylivingordeadorganism.Commonlyusedsourcesfor
DNA isolationare whole blood,hair,sperm, bones,nails,tissues,saliva, epithelialcells,urine, bacteria,
animal tissues,andplants.
The traditional methodsforthe isolationof DNA are more time consumingandthe reagentsusedare
costly.A fewmodificationsinthisprotocol make itsimple toprepare PCR-qualitygenomicDNA than
traditional methods.Inthismodifiedmethod(HotSHOTmethod),samplesare incubatedbrieflyinhot
NaOH andthenneutralizedbyTrisbuffer.Thismethodfoundtobe rapid,reliable,andinexpensive for
the isolationof PCR-qualityDNA fromzebrafish tissues.These advantagesmake ituseful forhigh-
throughput applications.Althoughitisaveryrapidand simple method,the qualityof DNA isnot
adequate forall applications.ItisfoundthatDNA obtainedfromthismethodisshearedandits
concentrationisveryless.The DNA isextensivelyshearedanditsconcentrationistoolow.Itisalso
possible thatthe DNA wasnot efficientlydigestedbythe restrictionenzyme becauseof interfering
substancesinthe DNA preparationorbecause the DNA didnot reanneal efficientlyafteritwas
neutralized.SothismethodismainlyforPCRrelatedapplications.AlthoughHotSHOTDNA preparation
islikelytobe limitedtoPCRapplications,the savingsintime andreagentsovertraditional DNA
preparationmethodsis substantial,andthe qualityof the resultsisasgoodas or slightlybetterthan
DNA preparedbytraditional methods.
Principle
The isolationof DNA usuallybeginswithlysisof tissue orcellswhichisessential forthe destructionof
proteinstructuresandalsoallowsthe release of nucleicacidsfromthe nucleus.Sodiumhydroxideis
mostlyusedtoextractDNA outof a cell.Ithelpsto breakdownthe cell wall bylooseningthe hard
structure of a cell wall ormembrane.More importantly,NaOHdisrupts the hydrogenbondingbetween
the Nitrogenbasesand convertingthe double-strandedDNA (dsDNA) (includingthe genomicDNA
(gDNA) andthe plasmid) tosingle strandedDNA (ssDNA).Thisprocessiscalleddenaturationandisthe
central part of thisDNA extractionprocedure.Presenceof sodiumhydroxidemakesthe solutionvery

3. basicor alkaline.Tris-Cl maintainsthe pHof the solution.Basicallyitreactswiththe lipopolysaccharides
presentonthe outermembrane whichmake the membrane permeable
Requirements
 Fish fillet
 Micropipette and pipette tips
 Scalpel
 Micro centrifuge tubes
 Mortar and pestle
 Centrifuge
 Water bath
 4o
c freezer
Reagents
 50mM NaOH solution
 1M Tris-Cl
Procedure
 Pluck out 20-100 mg of fish fin using a sterile scalpel and a forceps.
 Homogenize the fins using sterile mortar and pestle.
 Aliquot 100 μl of 50 mM NaOH in to a microfuge tube.
 Transfer the homogenized fins of zebra fish into this micro centrifuge tube.
 Incubate at 95°C for 20min in a water bath.
 After the incubation, the tubes are allowed to cool at 4°C.
 Add 1/10th volume of 1 MTris-HCl (pH 8.0) for neutralizing the basic solution.
 Centrifuge the sample at 5000rpm for 10min to pellet the debris. The supernatant obtained is
collected in afresh micro centrifuge tube (1-5μl of this supernatant can be used immediately
per 25 μL of PCR reaction mixture).
 Store the sample for at least 3 months at 4°C or longer at -20°C freezer.
Observationsand result
-The exactpH is maintainedby- Tris-Cl.
-Denaturation isthe central partof thismethod.

4. -95°C is the incubationtemperatureforDenaturation.
-Neutralization isthe role of Tris-Cl.
-Cell denaturationisdone by- NaOH,itlyse cell toexpel nucleicmaterial,anddisrupt
nitrogenousbases.
DISCUSSIONAND CONCLUSION
Deoxyribonucleicacid (DNA) extractionisthe processbywhichDNA isseparatedfromproteins,
membranes,andothercellularmaterial containedinthe cell fromwhichitisrecovered .
Preparation DNA forPCR iscostlyand laborious,primarily becauseof the difficultyin physically
separatingDNA fromothertissue components.Thislevel of purification isunnecessaryformanyPCR
applications.A simplealternativeinthismethod,lysisinanalkaline reagent andneutralization witha
suitable buffer,hasbeen usedtoprepare genomicDNA from buccal swabs,whole blood,semenand
forensicsamplescollectedfromhumans.
A fewmodificationsof this methodmake itpossibletoprepare PCR-qualityfishfillet genomicDNA with
a brief incubationinhotsodium hydroxide andpHadjustmentwitha Trissolution(HotSHOT).The Hot-
SHOT methodisrapid,inexpensive andmaybe carriedoutin 96-well plates,makingitamenable to
automationandhigh-throughputgenotyping. The reagentsforHot SHOT DNA preparationare simple to
prepare.An alkaline lysisreagentwith25mM NaOH, 0.2 mMdisodiumEDTA anda pH of 12 isprepared
by dissolvingthe saltsinwaterwithoutadjustingthe pH. A neutralizingreagentwith40mM Tris-HCl and
a pH of 5 ispreparedby dissolvingTris-HCl(notTrisbase) in waterwithoutadjustingthe pH.Tissue
samplesare collectedintoa96-well thermal cyclerplate orthermal cyclerstriptubes.The tissue sample
mustbe small;toolarge a sample can cause the methodto fail.Alkaline lysis reagent(75mLisaddedto
the samples andheatedto95°C for 10 minto 1 h. The undissolvedtissuedoesnotinterfere withPCR.
Afterheating,samples are cooledto4°C, and 75mL neutralizing reagentare addedtoeachsample. One
to five microlitersof the final preparationare usedpereach10-mLPCR volume.The combinationof the
alkaline lysisandneutralizingreagents yieldsabufferconsistingof 20 mM Tris-HCl (pH8.1) and 0.1 mM
EDTA, whichissimilartoa common DNA storage buffer.
The DNA extractionprocessrequirescareful handlingof biological materialtopreventsample
contaminationandcrossover.Tubesshouldbe carefullylabeled,especiallywhentransfersare required.
Robotsmay be employedtoextractreference samplesandsome evidence samples,butother
evidentiarysamplesmayrequire the directattentionof aDNA analyst.

5. REFERENCE
 Preparationof PCR- Quality Mouse GenomicDNA withHotSodiumHydroxide andTris
(HotSHOT) BioTechniques29:52-54 (July2000).
 G.E. Truett etal, BioTechniques29:52-54, 2000. Preparationof PCR-qualitymouse genomic
DNA withhot sodiumhydroxide andtris(HotSHOT).
 Method forisolationof PCR-readygenomicDNA fromzebrafishtissuesNathanD.Meeker,
Sarah A.Hutchinson,LinhHo, andNikolausS.Trede HuntsmanCancerInstitute,Universityof
Utah, SaltLake City,UT, USA BioTechniques43:610-614 (November2007).
 DNA sequencingII:optimizingpreparationandcleanup,Volume2(textbook) ByJanKieleczawa
 http://www.ehow.com/about_6504902_sodium-used-dna-extraction_.html#ixzz1Wm2TNKmy