PRODUCT INFORMATION

Thermo Scientific GeneJET Gel Extraction Kit

K!"#$% K!"#&

www.thermoscientific.com/onebio

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Ex(ir) *ate + CERTIFICATE OF ANA',SIS

The kit was tested in the extraction of 100 bp DNA fragment from a 2% agarose gel and a 5 5 kb DNA fragments from 1% agarose gel according to the protocol described in the man!al The "!alit# of the extracted DNA was e$al!ated spectrophotometricall#% b# agarose gel electrophoresis% digestion with Thermo &cientific 'astDigest restriction en(#mes and a!tomated fl!orescent se"!encing -.alit) a.thori/e* 0)1 )!rgita *ilinskiene

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CONTENTS -./0.N1NT& .' T21 34T &T.+A51 AND &TA6474T8 D1&-+40T4.N 0+4N-4071 4/0.+TANT N.T1& ADD4T4.NA7 /AT1+4A7& AND 1:;40/1NT +1:;4+1D 0;+4'4-AT4.N 0+.T.-.7 T+.;671&2..T4N5 &A'1T8 4N'.+/AT4.N

page 2 2 2 9 9 9 < = ,

COMPONENTS OF T2E KIT GeneJET Gel Extraction Kit 6inding 6!ffer @ash 6!ffer AconcentratedB 1l!tion 6!ffer A10 m/ TrisC2-l% p2 , 5B 5ene)1T 0!rification -ol!mns Apreassembled with collection t!besB 3! (re(4 >30=?1 90 m7 ? m7 15 m7 50 &3! (re(4 >30=?2 150 m7 <5 m7 90 m7 250

STORAGE AND STA5I'IT, The Thermo &cientific 5ene)1T 5el 1xtraction 3it sho!ld be stored at room temperat!re A15C25D-B 'or col!mns we recommend <D- storage for periods greater than 1 #ear An# precipitate that forms in the b!ffers d!ring storage can be redissol$ed b# inc!bating briefl# at 9ED-% then cooling to room temperat!re before !se Note6 Clo4e the 0a7 8ith GeneJET P.rification Col.mn4 ti7htl) after each .4e9 DESCRIPTION F The 5ene)1T 5el 1xtraction 3it is designed for rapid and efficient p!rification of DNA fragments from standard or lowCmelting point agarose gels r!n in either TA1 or T61 b!ffer The kit !tili(es a proprietar# silicaCbased membrane technolog# in the form of a con$enient spin col!mn The kit can be !sed to p!rif# DNA fragments from 25 bp to 20 kb in si(e The reco$er# rates are !p to ?5% in a 100 bp G 10 kb DNA fragment si(e range Asee 'ig 1B 1ach 5ene)1T p!rification col!mn has a binding capacit# of !p to 25 Hg of DNA and can process !p to 1 g of agarose gel The entire proced!re takes I!st 15 min and the isolated DNA is read# to !se in all common downstream applications incl!ding ligation% restriction digestion% 0-+% se"!encing and labeling

Fi76 $6 +eco$er# dependence on DNA fragment si(e

PRINCIP'E The DNA fragment of interest is excised from an agarose gel% placed in a microcentrif!ge t!be% sol!bili(ed in binding b!ffer and applied to the col!mn The chaotropic agent in the binding b!ffer dissol$es agarose% denat!res proteins and promotes DNA binding to the silica membrane in the col!mn As an added con$enience% the binding b!ffer contains a color indicator that allows for eas# monitoring of the sol!tion p2 for optimal DNA binding 4mp!rities are remo$ed with a simple wash step 0!rified DNA is then el!ted from the col!mn with the el!tion b!ffer The reco$ered DNA is read# for !se in downstream applications IMPORTANT NOTES 0rior to the initial !se of the kit% dil!te the :a4h 5.ffer AconcentratedB with ethanol A?=C100%BJ 3! (re(4 &3! (re(4 >30=?1 >30=?2 @ash 6!ffer AconcentratedB ? m7 <5 m7 1thanol <5 m7 225 m7 Total Kol!me 5< m7 2E0 m7 After the ethanol has been added% mark the check box on the bottle to indicate the completed step 1xamine the 5in*in7 5.ffer for precipitates before each !se +eCdissol$e an# precipitate b# warming the sol!tion to 9ED- and cooling to 25D @ear glo$es when handling the 5in*in7 5.ffer as this sol!tion contains irritants Asee p E for &A'1T8 4N'.+/AT4.NB Do not re!se electrophoresis b!ffer when extracted DNA fragment will be !sed directl# for se"!encing

ADDITIONA' MATERIA'S AND E-UIPMENT RE-UIRED 1thanol ?=C100% 4sopropanol 9 / sodi!m acetate% p2 5 2 Ama# be necessar#B /icrocentrif!ge 1 5 or 2 m7 microcentrif!ge t!bes 2eating block or water bath

PURIFICATION PROTOCO' Note +ead 4/0.+TANT N.T1& on p 9 before starting All p!rification steps sho!ld be carried o!t at room tem(erat.re All centrif!gations sho!ld be carried o!t in a tableCtop microcentrif!ge at ;$&!!! 7 A10 000C1< 000 rpm% depending on the rotor t#peB Ste( Proce*.re 1xcise gel slice containing the DNA fragment !sing a clean scalpel or ra(or blade -!t as close to the DNA as possible to minimi(e the gel $ol!me 0lace the gel slice into a preCweighed 1 5 m7 t!be and weigh +ecord the weight of the gel slice
Note6 4f the p!rified fragment will be !sed for cloning reactions% a$oid damaging the DNA thro!gh ;K light expos!re /inimi(e ;K expos!re to a few seconds or keep the gel slice on a glass or plastic plate d!ring ;K ill!mination

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Add $1$ <ol.me of 5in*in7 5.ffer to the gel slice A$ol!meJ weightB Ae g % add 100 H7 of 6inding 6!ffer for e$er# 100 mg of agarose gelB
Note6 'or gels with an agarose content greater than 2%% add 2J1 $ol!mes of 6inding 6!ffer to the gel slice

4nc!bate the gel mixt!re at 3!>"!?C for $! min or !ntil the gel slice is completel# dissol$ed /ix the t!be b# in$ersion e$er# few min!tes to facilitate the melting process 1ns!re that the gel is completel# dissol$ed Kortex the gel mixt!re briefl# before loading on the col!mn -heck the color of the sol!tion A #ellow color indicates an optimal p2 for DNA binding 4f the color of the sol!tion is orange or $iolet% add 10 H7 of 9 / sodi!m acetate% p2 5 2 sol!tion and mix The color of the mix will become #ellow Optional: !se this step onl# when DNA fragment is L500 bp or M10 kb long 4f the DNA fragment is L500 bp% add a 1J2 $ol!me of 100% isopropanol to the sol!bili(ed gel sol!tion Ae g 100 H7 of isopropanol sho!ld be added to 100 mg gel slice sol!bili(ed in 100 H7 of 6inding 6!fferB /ix thoro!ghl# 4f the DNA fragment is M10 kb% add a 1J2 $ol!me of water to the sol!bili(ed gel sol!tion Ae g 100 H7 of water sho!ld be added to 100 mg gel slice sol!bili(ed in 100 H7 of 6inding 6!fferB /ix thoro!ghl# Transfer !p to ,00 H7 of the sol!bili(ed gel sol!tion Afrom step 9 or <B to the 5ene)1T p!rification col!mn -entrif!ge for 1 min Discard the flowCthro!gh and place the col!mn back into the same collection t!be

@ for L500 bp and M10 kb DNA fragments

Note6 4f the total $ol!me exceeds ,00 H7% the sol!tion can be added to the col!mn in stages After each application% centrif!ge the col!mn for 90C=0 s and discard the flowCthro!gh after each spin +epeat !ntil the entire $ol!me has been applied to the col!mn membrane Do not exceed 1 g of total agarose gel per col!mn -lose the bag with 5ene)1T 0!rification -ol!mns tightl# after each !seN

Ste(

Proce*.re Optional: !se this additional binding step onl# if the p!rified DNA will be !sed for se"!encing Add $!! A' of 5in*in7 5.ffer to the 5ene)1T p!rification col!mn -entrif!ge for 1 min Discard the flowCthro!gh and place the col!mn back into the same collection t!be Add B!! A' of :a4h 5.ffer Adil!ted with ethanol as described on p 9B to the 5ene)1T p!rification col!mn -entrif!ge for 1 min Discard the flowCthro!gh and place the col!mn back into the same collection t!be -entrif!ge the empt# 5ene)1T p!rification col!mn for an additional 1 min to completel# remo$e resid!al wash b!ffer
Note6 This step is essential to a$oid resid!al ethanol in the p!rified DNA sol!tion The presence of ethanol in the DNA sample ma# inhibit downstream en(#matic reactions

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Transfer the 5ene)1T p!rification col!mn into a clean 1 5 m7 microcentrif!ge t!be Anot incl!dedB Add 3! A' of El.tion 5.ffer to the center of the p!rification col!mn membrane -entrif!ge for 1 min #
Note 'or low DNA amo!nts the el!tion $ol!mes can be red!ced to increase DNA concentration An el!tion $ol!me between 20C50 H7 does not significantl# red!ce the DNA #ield 2owe$er% el!tion $ol!mes less than 10 H7 are not recommended 4f DNA fragment is M10 kb% prewarm 1l!tion 6!ffer to =5D- before appl#ing to col!mn 4f the el!tion $ol!me is 10 H7 and DNA amo!nt is L5 Hg% inc!bate col!mn for 1 min at room temperat!re before centrif!gation

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Discard the 5ene)1T p!rification col!mn and store the p!rified DNA at C20D-

TROU5'ES2OOTING Pro0lem 'o8 DNA )iel* Po44i0le Ca.4e an* Sol.tion Incom(lete 4ol.0ili/ation of the 7el 4lice Kerif# that a 1J1 $ol!me of 6inding 6!ffer is added to a precisel# weighted gel slice Ae g for e$er# 100 mg of agarose gel% add 100 H7 of 6inding 6!fferB 1ns!re that the gel slice is completel# dissol$ed before appl#ing to the 5ene)1T p!rification col!mn A large amo!nt of agarose or a gel slice with an agarose percentage greater than 2% ma# re"!ire extra time to dissol$e 4n some cases larger $ol!mes of 6inding 6!ffer and additional $ortexing of the gel sol!tion facilitate sol!bili(ation Inefficient DNA 0in*in7 -heck the color of the sol!tion after the gel slice is completel# dissol$ed A #ellow color indicates an optimal p2 for DNA binding 4f the sol!tion color is orange or $iolet% add 10 H7 of 9 / sodi!m acetate% p2 5 2 sol!tion and mix The color of the mix will become #ellow Inefficient mem0rane 8a4h 1ns!re that recommended $ol!me of ethanol was added to the @ash 6!ffer AconcentratedB prior first !se Asee p 9B Inefficient DNA el.tion Add the 1l!tion 6!ffer directl# to the center of the membrane and not to the side of the 5ene)1T p!rification col!mn ;se 20C50 H7 of 1l!tion 6!ffer and ens!re that the $ol!me completel# co$ers the s!rface of the membrane 4ncrease the 1l!tion 6!ffer $ol!me twice or perform two el!tion c#cles when p!rif#ing larger amo!nts of DNA Ae g M15 HgB 4n step ,% ens!re all resid!al wash b!ffer is remo$ed from the membrane 7onger centrif!gation time Aextra min!teB can aid in remo$al of wash b!ffer DNA *oe4 not remain in an a7aro4e 7el 8ell Pre4ence of re4i*.al ethanol 4n step ,% ens!re all resid!al wash b!ffer is remo$ed from the membrane 7onger centrif!gation time can aid in remo$al of wash b!ffer Contamination from re.4e* electro(hore4i4 0.ffer 4f extracted DNA will be !sed directl# for se"!encing% freshl# prepared electrophoresis b!ffers sho!ld be !sed both for gel preparation and for gel r!nning

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Pro0lem Do8n4tream a((lication4 are .n4.cce44f.l

Po44i0le Ca.4e an* Sol.tion Pre4ence of re4i*.al ethanol 4n step ,% ens!re all resid!al wash b!ffer is remo$ed from the membrane A prolonged centrif!gation time can aid in remo$al of wash b!ffer Inefficient mem0rane 8a4h 4f the collection t!be is o$erfilled d!ring the wash step% some of the wash b!ffer ma# remain in the bottom of the 5ene)1T p!rification col!mn To a$oid this% alwa#s discard the flowCthro!gh after centrif!gation El.ate contaminate* 8ith a7aro4e 1ns!re the gel slice is properl# sol!bili(ed d!ring steps 1C< Kerif# that a 1J1 $ol!me of 6inding 6!ffer was added to a precisel# weighted gel slice 7arge amo!nts of agarose or agarose gel percentages greater than 2% ma# take more time to dissol$e 4n some cases adding a larger $ol!me of 6inding 6!ffer and $ortexing the gel sol!tion more fre"!entl# can facilitate sol!bili(ation El.ate contaminate* 8ith exce44 4alt 1ns!re that the wash in step E is effecit$e 4nc!bate the 5ene)1T p!rification col!mn with the @ash 6!ffer for se$eral min!tes before proceeding to centrif!gation

Reference4 1 Kogelstein% 6 and 5illespie% D % 0reparati$e and anal#tical p!rification of DNA from agarose% 0roc Natl Acad &ci ;&A% E=% =15C=1?% 1?E? 2 /arko% / A % -hipperfield% + and 6irnboim% 2 - % A proced!re for the largeCscale isolation of highl# p!rified plasmid DNA !sing alkaline extraction and binding to glass powder% Anal 6iochem % 121% 9,2C9,E% 1?,2 9 6oom% + % &ol% - ) A % et al % +apid and simple method for p!rification of n!cleic acids% ) -lin /icrobiol % /ar% <?5C509% 1??0

SAFETY INFORMATION
5in*in7 5.ffer 2a(ardCdetermining component of labelingJ 7.ani*ini.m thioc)anate En 2armf!l Ri4F (hra4e4 +20O21O22 2armf!l b# inhalation% in contact with skin and if swallowed +92 -ontact with acids liberates $er# toxic gas +52O59 2armf!l to a"!atic organisms% ma# ca!se longCterm ad$erse effects in the a"!atic en$ironment Safet) (hra4e4 &? 3eep container in a wellC$entilated place &29 Do not breathe gasOf!mesO$apo!rOspra# &9=O9E @ear s!itable protecti$e clothing and glo$es &=0 This material and its container m!st be disposed of as ha(ardo!s waste &=1 A$oid release to the en$ironment +efer to special instr!ctionsOsafet# data sheets

PRODUCT USE 'IMITATION This prod!ct is de$eloped% designed and sold excl!si$el# for research p!rposes and in vitro !se onl# The prod!ct was not tested for !se in diagnostics or for dr!g de$elopment% nor is it s!itable for administration to h!mans or animals 0lease refer to www thermoscientific comOonebio for /aterial &afet# Data &heet of the prod!ct

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