ISOLASI DNA

http://www.le.ac.uk/biology/phh4/methods/dnarna.htm DNA and RNA EXTRACTIONS DNA and RNA EXTRACTIONS A protocol / method / schedule /procedure for extraction / isolation of both DNA and RNA from the same material typically plant leaf / lea es !"ee also DNA #solation protocol$ %$ &ake one medium si'ed leaf or half a large leaf !( to )* cm+)$, weigh and free'e in li-uid nitrogen. )$ )$ .rind the tissue in a bleached and baked pestle and mortar with li-uid nitrogen. /$ /$ &ransfer the powder produced to a l(ml 0alcon blue cap tube. Add ) ml of homogeni'ation buffer !4 ml per g$ and disperse the tissue in it. 4$ 4$ 1ea e for %* min at room temperature. ($ ($ Add ) ml of phenol/chloroform and ortex. 2R, %$ 3ut a small or medium si'ed leaf into a 44 x 54 (** guage !thick6$ plastic bag. )$ Add )7/ ml homogeni'ation buffer !you may need more for large lea es$ to the bag. /$ .rind the leaf inside the bag using the top end of a (* ml corex tube. 4$ 3our the homogenate immediately into a %( ml 0alcon blue cap tube containing e-ual olume of phenol/chloroform then ortex. ($ .oto step 5 5$ "pin for %* min at ),(** rpm in a bench centrifuge. 8$ &ransfer *.8 ml of the a-ueous phase to an 9ppendorf tube, add *.8 ml phenol/chloroform, ortex and spin for ( min. :$ &ransfer *.5 ml of the a-ueous phase to a fresh 9ppendorf tube, add *.5 ml phenol/chloroform, ortex and spin for ( min. ;$ &ransfer *.( ml of the a-ueous phase to a fresh 9ppendorf tube, add *.( ml chloroform/isoamyl alcohol, ortex and spin for ( min. %*$ Add 4< 1i=l to the final conc. of )< and lea e for se eral hrs at 4o= !better o/n$. "pin for %*7%( min. RNA should be in a pellet. Remo e supernatant and precipitate the DNA with ethanol. %%$ &reat RNA fraction with DNase RNase7free, and the DNA fraction with RNase7free of DNase. Notes: >omogeni'ation buffer: *.% < Na=l )? "D" (*m< &ris7>=l p> ;.* %* m< 9D&A !ideally D93= treated water and e erything else RNase free$ then add *.% mg/ml proteinase @ A added fresh B

3at >eslop7>arrison Cni ersity of 1eicester December )**/. !#Dm afraid # did not note the source of this protocol. Alex Eershinin first used for oil palm DNA and RNA extraction to look at retroelement acti ity 7 see @ubis "9, =astilho A<<0, Eershinin AE, >eslop7>arrison F". )**/. Retroelements, transposons and methylation status in the genome of oil palm !9laeis guineensis $ and the relationship to somaclonal ariation. 3lant <olecular Giology () : 5;78;. doi:%*.%*)//A:%*)/;4)/*;*;)$

DNA isolation & extraction CTAB TECHNIQUE / et!od / Sc!ed"le / #rotocol $OR DNA ISOLATION / DNA EXTRACTION $RO #LANT LEA$ / LEA%ES SA #LES &see also DNA RNA do"'le isolation (roced"re i) 'ot! DNA and RNA are needed* Reagents needed =&AG buffer )? =&AG )*gm =&AG )*m< 9D&A 4*ml 9D&A stock !*.(<$ %**m< &ris7=l p> :.* %**ml &ris7=l stock !%<$ %.4< Na=l ):*ml Na=l stock !(<$ make up to % 1itre with water, p> 8.( 7 :.*, and autocla e H *.)? <ercaptoethanol Iash Guffer 85? 9thanol %*m< N> 4 Ac DNA 9xtraction %. 3reheat (ml =&AG !add %*Jl mercaptoethanol to each (ml =&AG$ in a blue7topped (*ml centrifuge tube at 5*75( o =. Remo e and discard midribs, and wrap laminae in aluminium foil and free'e in li-uid nitrogen. *.( K %.* gm tissue/(ml =&AG !=an store leaf material after li-uid Nitrogen K %7) days at K)* or K:* for longer periods$ ). .ently crumble leaf tissue o er cold pestle of li-uid nitrogen. .rind fro'en leaf with one spatula of fine sand add *.( spatula of 3E33 powder after grinding. /. "crape powder into dry tube and add pre7heated buffer and mix gently. A oid lea ing dry material around rim of tube. AdLust =&AG olume to gi e a slurry7like consistency, mix occasionally. 4. #ncubate for 5* min at 5* o = (. Add e-ual olume of chloroform/iso7amyl alcohol !)4:%$, <ix for about /min, then transfer contents to narrow bore centrifuge tubes. Galance by adding extra chlor/iso. "pin (,***rpm for %*min !ensure correct tubes used$, brake off. !0or extra pure DNA isolation 7 spin and retain supernatant before chloroform extraction$. 5. Remo e supernatant with wide7bore pastette !cut off blue tip$ to clean tube, repeat chloroform extraction once. "upernatant should be clear, though may be coloured. 8. 3recipitate DNA with *.55 ol. of cold isopropanol 7 can lea e o ernight. "pool out or spin down DNA, )min at ),***rpm. :. &ransfer to (ml wash buffer for )*min. ;. Dry briefly and resuspend in %ml &.9. !can be left o ernight$

%*. Add %Jl %*mg/ml RNAse to each %ml &.9./DNA mixture and incubate for 5*min at /8 o =. !#f RNase in the sample doesnDt matter K stages %% and %) may be omitted$ %%. Dilute with ) olumes &9 and add *./ ol /< "odium acetate !p> :$ H ).( ol cold %**? ethanol, %). "pool DNA out. Air dry and resuspend in *.( to %ml &9 or water !takes time$ and free'e until re-uired. DNA Q"anti)ication An approximate way to determine DNA concentration is to look at the iscosity of the solution: not accurate to %*? but, unlike spectrophotometry, you will not get results which are %* or %** times wrong6 #n a microcentrifuge tube, DNA solutions stronger than *.% ug/ul will show a reluctance to pour when you tilt the tube. 0rom about *.( ul/ug and abo e, you can tilt the tube 7 ery gently 7 and the solution will stay at the end. #f you dip a %*7)** ul !yellow$ pipette tip into the solution and pull it away, a solution of %ug/ul will form a distinct string from the surface to the tip which breaks when about % to ) mm long. <ake a *.:? agarose gel with %x &A9 and *.%Jl of 9thidium bromide !%*mg/ml$ per %*ml solution. 1oad samples undiluted and at a % in %* !%H;$ dilution., with /Jl loading buffer. Also include a 1amda ladder cut with >ind### and 9coR#. &his contains %**ng of DNA per microlitre and use as follows: %Jl ladder H 4Jl water H )Jl loading buffer )Jl ladder H /Jl water H )Jl loading buffer &he different bands of the ladder are of known molecular weight and known DNA concentration. <atch the brightness of your samples with those of the two dilutions of the ladder. Refer to the diagram to match the band with the concentration. Remember that although the ladder concentrations are absolute, you ha e loaded (Jl of sample and also diluted some of them. &his must be taken into account when calculating the strength of the sample s in ng/Jl. 3estles and mortars washed for )*7/*min in *.)(< >=l, rinsed in water and air7dried, all mess to be tidied up and tubes washed and left to drain. 3at >eslop7>arrison, &rude "chwar'acher, Fohn Gailey Cni ersity of 1eicester )**//)**;. "ee www.methods.molcyt.com

A #LANT NUCLEAR DNA #RE#ARATION #ROCEDURE

3<G Newsletter !%;:%$ Eol. ## F. Gedbrook, ="#R2 Di ision of 3lant #ndustry, 32 Gox %5**, =anberra =ity A=& )5*%, AC"&RA1#A &his method can be used to prepare 3etunia c . Rose du =iel nuclear DNA suitable for restriction en'yme analysis and cloning. =hloroplast DNA contamination will ary depending on the genotype. .reater chloroplast DNA contamination results when this method is used with the 4<itchell4 line of 3etunia. %. Moung lea es !)* g$ from %7/ month old 3etunia plants are ground at 4 = with a minimal olume of *./ < sucrose, ( m< <g=l), (* m< &ris >=l !p> :$ !>G$ in a mortar and pestle. &he slurry olume is brought to %** ml with >G and filtered through ) layers, then 4 layers of cheesecloth. ). &he filtrate is centrifuged at %*** g for ( min and the pellet resuspended in %** ml of >G and repelleted at %*** g for ( min. &his pellet is resuspended in %** ml of >G containing )? &riton N7%** incubated at 4 = for %* min and centrifuged at %*** g for ( min. &he pellet is washed with %** ml of >G with &riton N%**, recentrifuged, and the pellet cleaned of excess li-uid with a paper towel. /. &he pellet is resuspended in %5 ml of /* m< &ris7>=l !p> :$, %* m< 9D&A !RG$, and transferred to a %** ml flask. ) ml of %*? !w/ $ "arkosyl is added along with ) ml of ( mg/ml pronase. &he mixture is heated at 5* = for ( min and incubated with gentle shaking at /8 = for (7%* hrs. &he solution is highly iscous and contains among other things, starch grains and nuclear debris. 4. After the incubation, measure the total olume of the lysate and bring it to a olume of )* ml, Add )* g of =s=l and dissol e completely o er a period of about two hours at 4 =. (. Gring the mixture to room temperature, add ) ml ethidium bromide !%* mg/ml$. mix in gently and thoroughly. 5. Dispense in centrifuge tubes and centrifuge at /(@ rpm for /* hrs at %( =. =ollect the DNA band under long wa e u light. 8. Recentrifuge the DNA and collect, extract the ethidium bromide with RG7saturated isoamyl alcohol and dialy'e against ( m< &ris7>=l !p> :$, *.)( m< 9D&A. "tore at 4 =.

#LANT DNA ISOLATION

%. 3repare (7)* g of clean, fro'en, young lea es taken from plants grown under controlled conditions and exposed to darkness for two days prior to isolation. Remo e mid7ribs. ). .rind lea es in stainless steel blender containing %(*7)** ml of ice cold > buffer at maximum speed for % min. /. 3our the homogenate in a )(* ml centrifuge bottle !on ice$ while filtering through one layer of miracloth !=albiochem$ under four layers of cheesecloth !all pre iously wetted with %* ml clod > buffer$. 4. =entrifuge at )*** g, 4 =, )* min. (. Discard the green supernatant and resuspend the pellet in 4* ml ice cold >& buffer. 5. &ransfer to a (* ml teflon tube !2akridge 2@$ and centrifuge at )*** g, 4 =, %* min. Repeat until pellet of nuclei becomes greyish7white !%7/N$. #f anthocyanins are present in the plant, the pellet will be reddish7brown. 8. Resuspend the pellet thoroughly in %) ml of >& buffer then add %) ml of lysis buffer. :. #mmediately, add )/.): g of powdered =s=l and incubate the tubes at ((75* = for one hour with occasional in ersion. ;. After solubili'ation of the =s=l, centrifuge the tubes at ):*** g, %( =, /* min. %*. 0ilter the supernatant through two layers of cheesecloth into a /: ml -uick7seal tube containing %.48 ml 9tGr solution using a (* ml syringe and a %5 . needle as a funnel. =omplete olume with =s=l solution. %%. DNA is reco ered after centrifugation using standard procedures. +X TE: %* m< &ris7>=l !p> :$, % m< 9D&A +X H: %*N >, 4** mlO sucrose, 5:4 gO P7mercaptoethanol, : mlO water to 4*** ml Qp> ;.(R L,sis '"))er: Na7sarcosine, 4 gO &ris base, ).4) gO Na)9D&A, ).;: gO water to )** ml Qp> ;.(R +-X H: spermidine, )*./( gO spermine, )8.: gO Na49D&A :/.)4 gO &ris base, )4.) gO @=l, %%;.) gO water to %;** ml Qp> ;.(RO Add 3henyl methyl sulfonyl fluoride !3<"0$ solution Q8 g in %** ml of ;(? ethanolR +X HT: %N >, %*** mlO &riton N%**, ( ml CsCl sol"tion: =s=l, ;8 gO %N &9, %** ml EtBr sol"tion: 9tGr, % gO water, %** ml