ixpt. No| GHT Qurne> Peiton Das Rd 210 - 03/74 - Leiaertien ee Homme oes al DWA isdation Som Sith Blead- | Reman | 2 | Rogrnce gel Checdnn phorasis (ae | Palyamennte Chaim Reason (Pcp) t= 3 4. If || | S-| RNA extanction funn tith blood. reNAL @o Date Aim DNA \saolation from firh Aolond. C Phenol chlowfmmn extraction onethod? | Priore! ple > quit —| igudd ate DA, RNA Phen! chiarfrem extortion method is a ti ection technique used lnmalecular biolag 1 nitive along AProtvin. Fis \solattont of ONH, the phemal elaxo SLD canye!~alleohol (as:2u:1) ce parte 4, onganle phox | comrtalat gt Lipid. carbahy dante ete. Sram the Oquedus fave Combatning the Dui, Phenal form a Suscy , se poxnted by the chimndarm while lsa-amy!odcalrol ack a8 on amtifooming agent The lyse tudfer (Teds cl, EDTH, SDs) diene! Ahe cell imbegrity while providing popes pl condiHone- and Safe 4 Lxom aul ease." | Fac Precipitation af PNA chilled ethonal i utileed Sollawed A Anashing with #5% ethanol 4o remove ary 4naces of pheongl. TE Houtles conlaining -triscl and Ed1a ic wtilined tn diccalve 4h lemecbpitated nwa ma suitable pH anedia protected dene pare Dye mganic place Material required axe — @ Proteimace 1a (20me /rnl) (2) Lysis bu Hew (100 oni) O) Trie-c\ G4 m\ (000 ™ NI pu: @)- Vamt Cleon) (oy) Epa = Goo mM, pH=g) = 20 mJ _Cypeanm) —_©@) S04 +» Soo m4 6-57) Teacher's Signatureodin dark los Lsornaryl alee Qsd_equal ya Teacher's Signature3 uibvifugation 6. |_omd keep int fresh © 9) Add Ao volume of am sodium accrtade . mic goratl ye |_\e) To se aqueous layer now pow coulle yolyme of hill 0. absolite tron! d dep the appendort tobe tyldtlys | wo femtattune the above salut ot lycoo RPM dow 20 at atc: 12) Decomt the alcolal cnompletely amd add dao Ubat 14 chilled et omal = Comtsiduge the above solubion of oro REM fax Sani ok uc. oi ac Me Decomt the oltohal completely and ale day the pcilet af it eo fempeacdure 15. Pal a Al ot Te lather, water oligfitly and keep _ Itetn tconm temp fr llowed tre eberage pid 4% | Rest > well iscloded pa is ohsenved. . Toacher's Signature.No. Page No 9 Date [Rim> Agarcose gel cleden wns) s« Prtarcl ple > a3 4 Agcanse gel ovhomsle is a onutinely use {method f : c > g d | fone tre parading. “DNA ox ie ermnted f Hoare om aloe by the aid af am clechde feild aleve aegitives F L charged ecules ¢ pl grote Agwpovds amade. “The anigraton Saw | ie debrmined ly the qnolecula welaht where somal welolct Lanale cule fynl qe whe tarte wan heavy onolecule. Im odition to sire separnton, muclic acid Gn hiomartin sing a0. atc Lgelelecterhorns!s com be tmitial strep foe feuther pur fica! an ExtenHon of the -beohmigue includes wv brans— Lluaainatlon “The agoovse’ ge elechn pms te widely weed do estima dhe sre of Dwi fragments adder diqe Ling with tcovdriet emoyme. H hne alco heen a ‘couwtin deal im anilec gous Meg lagmosts ox gemedic Finger grb ding vin amaly co PCR producl . Gepanation af esta ded genomic vA poloa— [to Souther Iotet and separation ef RNA gator ta mthern bof | Larce alen dependent on agarose gel clecbrophernels. To adilian to providing am excellent medium far an it store amalyes. }-ogounce gelallow purification of pwa droginents. Since [purification cf DWA Sroprnerd sive senombed in an cqqose Lgelis recsecomy fax sum ber amabe ctl Aechl qu reine | Coming. Tis vital do puridy Sveqone nts of Indeweet | Gorn the ge ‘om. Teacher's Signature.ppt. No. Page No. Nee, Dae * Sax muctetc acid @ddivon bromide CE* bs) is comanon di viowlootion - “Etbe te a Clue nd _die and 1} fortes date ff between mutete aed bares amd panvide ppartemit do cosih fetert mutlice acid fraqment-in gel under Uv Lluwina Hon Uy morescence Should be ands degrdes by long Soo -soo mm. Exposing DNA ta |p: formed scagidly 4 roureenule! lemposumes and thus the sharp ef the lands would be meqctively aterted. Most agmme gcl ome prepared ulla the agoame covcentrabton scomging £-%%.. roxlus loutfer ave need Smeg fecbsn-phoxos)s tina anost common futher tr au ic Quid ame Tals /Acetade | ZepTA (Trt) ond Tats /own rte /EntA (TRE) - Dui dongonerct Lomigantes with ditlered reates fm this two buffer due do Alte! lonte strength: Pu tfer mat only establivyh oof deal pH buf - Th Limovide lone do euppand oo nducttviby. “ry qemerul tre {deol Luter thould onduce tose beat, have along tide amd 0 qcad conduclivity. Molertals Re quived = ts ; @_ Agarose } (y 4x TEAR Gy) Adiwon bom de Ww) Porsafilon [efor tn end_pne sages |__vO DwA/ awe Soomple vo Uv- trons i Ila ination Teacher's SignatureNo. [PageNo. Date Preatocal L) To peepore +5 ¥ agamce gel fax genomic DIVE separ ond odd it +0 4o onl of £* THE eld 0.6 gan otegaxn Piston 2) Cover the’ opening af the comical flask with paper and Socom it ving cubhex Lond. 4) Bedore takinag-eudthe conleut Haske, allow Hre ala ubles tn dicappear. | 5) Remave the cmercomd allow the g tio. ae G) Bld 12 Ail of ¢Hnidiven mponide de i. a‘ D Crombly wotnte the conical Mask to ome dhe ethidium | tromide tra the q-el: pHs ef @) Pure the gel mba ges cortex Choving well-tomb otatthed do 4): Moke sive that alo buble are aot Smoned 9 Ailow the gel te solidid 10.) “Tn the ameomtime pour 1x TRE Aw Sea 4a th Lelectmphome< ludfer tank. Ai) fitter the gel thas calidisied arfutly rernove the AL} Caml ts form the wells. 12. Take out +h gel cording daroy ontalning 4he gel amd plore i+ in the buffer donk 4 J Bows sade UUTHE Wwiter tn male cure thul dhe | wells awe connpletely subs mgd P| Teacher's Signature$4) Cul a lenoth of povotitm and aflach 4 yeas the tow tex ton} 45) Ante Yaca_se onus stil of som@leaned G Miy the sumele with the (onda loudfen ¢ corfully so as to mob cane a tale 1% tod the teort ot the 16) Noad DWA ima web fon aetowence . 19 Conmed the amode ond catnd 4o the power saanple shni laafy oe) Sy doen fax de srequixed pettod of dime. 2 Afters Polymevase chain reachon @cr) eh mesose Cham teachn ax pen \s a provees Hut ulilioe uritied errymes Sax ama OWA or RWA Sequence. Hence it is somedione tlso arch a do_o-andle cular moValngmophy'. Tue Wcauly fi DER ta Haat tel fealty Nate morse dhok hue the abillly ta worl even ty exreanely Lonmplecated aniytuares in vib eoples of spexj fic PCR equines a template onmlecule Gg- pve er By ) ou wont to Mpy amd dw primar which help ta tnldiation Ppmmcess of copying . The primers ave nade US of Soy Ldedterent type af chemical en pound cotled mucleptide rene Hore mejor ebepsin PCR, which ore cepted! m30-40 Homes. This 1s done om an automated evel which Con heat and coal the tubes with ther oNion amix tune hn La vety ght time. ¢ LQ) Tnitial Denalucation of aut. Bedore the cp press amare Intn the ical pattern Lam initial denatuawtion of Dit templates is done at owe fo [2S aninutes depending am the length. of dhe tomslate stand that ontalid be the whole genome pwn camaplelesn re Teacher's Signature,Page No. No. Date @ Dematu Durcing the Demelueation, the double te single sthromded pnt, all enoyenwhic + jon at 94°C 3) Nmeling ot _ss*c The grimars ave giggling armmd, roused by the Ltwmine ancthon: todic bands ane cometantty formed amd | tmsoken etween the single stonndond pmax and the single dconded template . The sume dang ala ais replated eo treet in wal incre Teacher's SignaturePage No id nthe Hind (ole- The mewl garliesioed target avglon DNA err - 1. cds sfill present Od will re tutil the ond adthe reaction . However adden po few cycle, the mewty evnthesized wi Sragmerts quttclely estabtithed sof as dhe gredoninat template. cyiderare Ayoicalhy tcrpeted fax 4p Hone Ss whe The origi Com sidersaHton ~ @ Template pwr - ae ce Neovly omy chmuded anedhed ic suitable carr MwA Dudsi-fieatton. for adequate ama. f template DN Lis between 0.1 £ 4g fore genomic dive Sex a dada! | Lyreaction ovirdune of toe Ul. Loage ‘tomplal D Nil mmawels Losually imeates the yrild of mor specific PCR produel —_| ® Priner— eee = ) PCR Primers should be to24 oueleatides tar engl }—1) “The primers thauld vat be self complemendaye or Complementary to any other prioner in the oeacltan « Ag prevent paler — dime Shalepin dowonetion: | <0) GeC_ content solution te facilitate He ‘onanediafe ond amort e Sevtive Inhibition of | | Rwhose activity «fh ee eae | ae nized ow lysed | into We echays ageelomiata aire ase yee |Houmech ately along with phenal. Tt should be te comandeddhat dhe ptt of the Gumi dim thlocynate ebould toe acédit fox | [the total RNA tn be tocatized inthe Adquaus phase. Ow | | Oster balafton ts generally solubilioed im diedbyl pyromabanel| | ec) treated water rohted imechiae>-Qwiare tan voadee Land Jobawatory ytitises. Teacher's Signature| | Saas la= = [g)ttix S=6 Annes by inventing | [@) Incubate tor 15 oninude of Urs-34)!C. 5 # | } | fo) ConvbaiSneg of 12 000 REM For io aninute af atc. w Astes contsitug ator tremove the supewnatont completely. JZ [it eect Lhe jt a i aa of the tube. (ad {alas the RA pellet ace wlth Z5y. thane na ; wy) Contsifuge cof 2o00 open tax S unin at ht¢. (is) Discard all ethanol: e 5-10 on nutes