nila-pandhara padada

nila-pandhara padada heey eka tapasani tantra aahe jeey vector-aadharit aanvik cloning prayoganmadhye punarsanyojit jivanuncha jalad aanee soyiskar shodh ghenyas anumati dete. tapasanichi hii paddhat sahasa yogya jivanu taana vaparoon kelee jaate, parantu yistasarkhya itar jivanchahi vapar keela jaau shakato. dii. aine. aii. auph transformation
vectormadhye jodale jaate. tyanantar vectorla parivartanasathi vyavharya asalelya saksham yazman peshimadhye ghatale jaate, jeey nantar ex-galchya upasthitit vadhavale jaatat. punarsanyojit dna asalelya vahakanni rupantarit jhalelya peshi kahee vasahati tayar kartil; naun-abhikarmak plasmids rupantarit jhalelya peshi (i.eae. fakt vahak) nilya vasahatinmadhye vadhatat.
parshvabhoomi.
aanvik cloning hii aanvik jivashastrat sarvat samanyatah vaparlya jaanarya prakriyanpaiki eka aahe. ligeshandware plasmid vectormadhye swarasya asalele januk ghatale jaau shakate aanee plasmid nantar "escherichia kolai" peshinmadhye rupantarit kele jaate. tathapi, peshinmadhye rupantarit jhalelya serve plasmidmadhye ichchit januk samavisht asoo shakat nahee aanee pratyek swatantra vasahatimadhye samavisht kelelya vastuchi upasthiti tapasane velkhau aahe. tyamule, hii prakriya qamee vel aanee shram-gahan banavanyasathi, incert shodhanyachi eka paddhat upayukt tharel. antarbhusha shodhanyasathi vikasit kelelya suruvatichya paddhatinpaiki eka mhanje nila-pandhara padtalani, jee jivanunchya vasahatichya rangadvare cloning pratikriyanchya yashasvi utpadananchi olakh patavanyas anumati dete.
hii paddhat β-galactosidase janukachya α-purnatechya tattvavar aadharit aahe. α-purnatechi hii ghatana pratham franswa jacob aanee jacks monodchya prayogshalet agness ulamanane kelelya karyat darshavili gelee, jithe nishkriy utparivartak β-galactosidase karya hatavalelya kramane β-galactosidase tukadyane vachavale gele, jyamadhye toch kram, α-donor peptide, ajunahi abadhit aahe. langley "aanee itar."heey dakhavun dile qee utparivarti karyatmak nasalelya beeta-galactosidase aine-terminuschya kahee bhagat tyacha avashesh 11-41 kadhoon taknyat aala hota, parantu tyala beeta-galactosidase 3-90 avasheshanni tayar jhalelya peptideware purak kele jaau shakate. pahilya 145 amino esidsathi anukram coding asalele malika philaments fages nantar messing ett all yanni tayar kele.", aanee α-vectorchya vaparadvare purnatva nilya pattyanchya nirmitidvare darshavile gele jevha nishkriy prathine asalelya peshi fejdware sankramit jhalya aanee nantar ex-gall asalelya pattyanmadhye vadhalya.
viera aanee mesingdware plasmid cloning vectorchi pook malika malika pranalitoon vikasit kelee gelee hotee aanee yaa tapasani paddhaticha laabh ghenyasathi bandhanyat aalele pahile plasmid hote. yaa paddhatimadhye, plasmidmadhye jodale gelele dii. aine. aii. heey α peptidemadhye vyatyay aanate aanee tyamule purak prakriya viskalit hote aanee konatehi karyatmak β-galactosidase tayar houu shakat nahee. incert asalelya plasmids rupantarit jhalelya peshi kahee vasahati tayar karatat, tarr incert naan karata plasmids rupantarit jhalelya peshi nilya vasahati tayar karatat; asha prakare yashasvi bandhan parinam ayashasvi nilya vasahatinpasun tayar jhalelya peshinchya kahee rangane sahaj olakhala jaau shakato.
aanvik yantrana.
beeta-galactosidase heey 'lack' opronchya 'lacz' janukadware encode kelele prathin aahe aanee teey tyachya sakriy sthiteet homotetrammer mhanoon astitvat aahe. tathapi, 'eae' chya chya prabhedatoon vyutpann jhalela utparivartak beeta-galactosidase. kolaiche aine-terminal avashesh 11-41 kadhoon takale aahet aanee haah utparivartak, ω-peptide, tetramer tayar karnyas asamarth aahe aanee nishkriy aahe. prathinancha haah utparivartit prakar tathapi, prathinancha aine-terminal tukada, α-peptidechya upasthitit tyachya sakriy tetrameric sthiteet purnapane parat yeu shakato. α-peptideware utparivartak β-galactosidase karyachya bachawala α-complementation mhanatat. 
padtalanichya yaa paddhatimadhye, yazman 'eae. kolai strainmadhye 'lacz' hatvinara utparivartak utparivartak asato jyamadhye ω-peptide asato, tarr vaparlelya plasmidmadhye 'lacz' kram asato joou β-galactocydes, α-peptidechya pahilya 59 avasheshanna encode karato. donhihi swatach karya karat naheet. tathapi, jevha done peptides ekatra vyakt kele jaatat, jevha 'lacz' kram asalelya plasmidche plasmidche peshinmadhye rupantar hote, tevha teey eka karyatmak beeta-galactosidase enzyme tayar karatat.
nilee-pandhari tapasani paddhat yaa α-purnatva prakriyet vyatyay aanoon karya karate. plasmid "lacz" kramamadhye antargat ekadhik cloning site (emm. see. ess.) vahoon netat. lacz" kramatil heey emm. see. nirbandhit enzymesdware kapale jaau shakatat jenekarun "lacz" α janukamadhye bahya dii. aine. aii. samavisht kele jaau shakate, jyamule α-peptide tayar tayar janukat vyatyay yeto. parinami, inserts plasmid asalelya peshinmadhye, konatehi karyatmak β-galactosidase tayar houu shakat nahee.
sakriy beeta-galactosidase upasthiti ex-galdware shodhali jaau shakate, jee lactosechi rangheen pratirup aahe jee beeta-galactosidesdware 5-bromo-4-chloro-indoxyl tayar karnyasathi vibhajit kelee jaau shakate, jee nantar utsfurtapane diamerise hote aanee 5,5 '-diabromo-44'-dichloro-indigo haah chamakdar nila adravya rangdravya tayar karnyasathi auxidise hote. yamule karyatmak β-galactosidase asalelya peshinmadhye vaishishtyapurn nila rung yeto. tyamule nilya vasahati darshavtat qee tyanchyat akhandit 'lacz' asalela vector asoo shakato (mhanoon tyat bharr ghalata yet nahee), tarr kahee vasahati, jethe ex-gall hydrolyse kelele nasate, tyaa 'lacz' madhye ghalanyachi upasthiti darshavtat jyamule sakriy β-galactosidase nirmitimadhye vyatyay yeto.
rupantarit vasahatinmadhun thodya pramanat plasmid dna vegale karoon aanee shuddha karoon punarsanyojit pratirupanche pudhil vishleshan kele jaau shakate aanee pratirup kapanyasathi aanee tyat swarasyapoorn tukada aahe qaa heey nirdharit karnyasathi nirbandhit enzymescha vapar keela jaau shakato. dii. aine. aii. chee kramvari lavane aavashyak asalyas, vasahatinmadhil plasmid eka tappyavar vegale karane aavashyak aahe, mug teey nirbandhit enzymescha vapar karoon kapayache asso kinwa itar parikshane karayachi.
vyavharik vichar.
vichar padadyache niyojan kartana yogya prakarchya vahak aanee saksham peshi heey mahattvache aahet. plasmidmadhye 'lacz' α asane aavashyak aahe aanee asha plasmidchi udaharane mhanje mhanje aanee piebluescript. eae. kolai peshimadhye hatavalelya kramane (i.eae. (i.eae, aanee asha genotype asalelya samanyatah vaparlya jaanarya kahee peshi mhanje mhanje, jm109 aanee aanee. heey dekhil samajoon ghetale pahije qee glukojchya upasthitimule lack aperon prabhavit hoto. glukojchya aayatit guntalele prathine aay. aay. aii. jee. ell. see., glucose peshimadhye vahoon nele jaat asatana lactose pargaman bund karate. tyamule aggar platemadhye vaparlya vaparlya madhyamanmadhye glukojcha samavesh nasava.
ex-gell haah prakash-sanvedanshil asato aanee mhanoon tyache dravan aanee ex-gell asalelya plates andharat sathavalya pahijet. isopropyl β-d-1-theogalectopyrenoside (aay. pii. tii. jee.), jeey "laakh" opronche prerak mhanoon karya karate, teey laxchi abhivyakti vadhavanyasathi madhyamanmadhye vaparale jaau shakate.
ex-gell hii eka mahagadi samagri aahe, tyamule jivanunchi tapasani karnyasathi itar paddhati vikasit kelya gelya aahet. jivanunna padadyavar aananyas madat karnyasathi jee. eff. pii. haah paryay vikasit karnyat aala aahe. hii sankalpana α-purnatesarkhich aahe jyamadhye dii. aine. aii. samavisht kelyane vectormadhil coding kram viskalit houu shakato aanee asha prakare jee. eff. pii. utpadanat vyatyay yeu shakato jyacha parinam naun-fluorosing jivanumadhye hoto. jyaa jivanunmadhye punarsanyojak vahak (vector + incert) asatat, teey pandhare asateel aanee jee. eff. pii. prathine vyakt karnar naheet, tarr naun-rekinant (vector), yuu. vhee. prakashakhali ichchhashakti aanee fluoreses kartil. jee. eff. pii. chaa vapar sarvasadharanpane eka reporter januk mhanoon keela jaato, jithe vyakti nishchitpane tharvu shakatat qee clonemadhye sanshodhak vishleshan kareet asalele januk aahe qee nahee. kahee prasangi, jyaa madhyamat vasahati vadhatat teey padadyavar prabhav takoo shakatat aanee chukiche-sakaratmak parinam deu shakatat. madhyamavaril ex-gell kadhikadhi nila rung tayar karnyasathi qamee houu shakato kinwa jee. eff. pii. madhyamamule tyachi pratidipti gamavoo shakato aanee ichcha punarsanyojkansah vasahati nishchit karnyachya sanshodhakanchya kshamatevar aanee jyanchyakade tee nahee tyanchyavar parinam karoo shakato.
tote.
kahee kahee vasahatinmadhye anek karananmule ichchit punarsanyojit plasmid asoo shakat nahee. legated dna yogya asoo shakat nahee kinwa yogyaritya legated nasel, aanee kahee rekheey vectorche rupantar karane, tyachya tokanchi "durusti" karane aanee ekatra jodane shakya aahe jenekarun konatehi laxa tayar honar nahee aanee nilya vasahati tayar houu shakat naheet. utparivartanamule α-tukada vyakt naan honyas dekhil karanibhoot tharoo shakato. konatihi vahak nasaleli vasahat dekhil pandhari disel aanee vaparleli pratijaivik sampalyanantar kadhikadhi upgrah vasahati mhanoon disu shakate. nilya vasahatinmadhye incert asanyachihi shakyata aahe. jevha incert laxa janukasah 'framemadhe' asato aanee insertmadhye stop coden anupasthit asato tevha heey ghadate. jarr tyachi rachana viskalit jhaali nahee tarr yamule fusion prathinanchi abhivyakti houu shakate jyamadhye karyatmak lacz α aahe. yogya punarsanyojakachi rachana kahivela halakya nilya vasahati deu shakate jyamule tyachi olakh guntaguntichi houu shakate.

