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[求助]
簡(jiǎn)并引物克隆中的一些問題
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我設(shè)計(jì)了一對(duì)兼并引物,想從未知基因組中克隆出一個(gè)酰胺酶基因片段。BLAST以后發(fā)現(xiàn)和大豆慢生根瘤菌的酰胺酶的序列有99%的相似性,而且我們實(shí)驗(yàn)室也在做這種大豆慢生根瘤菌的酰胺酶。起初懷疑是染菌了,又重新克隆了一次(很小心,避免染菌)可測(cè)序結(jié)果還是一樣的。 所以我懷疑是我這種菌的基因組被污染了,請(qǐng)問基因組會(huì)這么容易被污染嗎?而且自從我們實(shí)驗(yàn)室有了大豆慢生根瘤菌后,我這種菌的基因組再也沒有用過,這是第一次用。請(qǐng)高人指點(diǎn)。 |

鐵蟲 (初入文壇)

鐵桿木蟲 (著名寫手)

鐵桿木蟲 (著名寫手)
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前輩:您好。之前在論壇上看到您給一些蟲友在設(shè)計(jì)兼并引物方面提出了很多建議,請(qǐng)問您能否幫我設(shè)計(jì)一下(自己之前試著設(shè)計(jì)了一下,效果都不好),所以想請(qǐng)教高手。 我需要克隆一個(gè)酰胺酶,目前知道能催化我的底物的菌種的酰胺酶有兩種(下述A,B),但導(dǎo)師讓我做的菌種中有該酶的活性,但是這種菌的全基因組不知道,與A,B的同源性相差甚遠(yuǎn)。以下C,D,E,F,G••••••是我根據(jù)A為模版blast后的酰胺酶序列,從中挑選的與我手中的菌都屬于同一綱的菌,同源性從高到低排列的,但和A的相似性很低。 下面我把這些菌的酰胺酶基因上傳上去,能否花費(fèi)您寫時(shí)間幫我看看,因?yàn)橹霸O(shè)計(jì)了幾次,效果不是很好。 >A: 1 mgiklptled lreiskqfnl dledeelksf lqllklqles yerldslpdy tprvkyqrtl 61 grppmkeenp ygalvwitsi kgkeegklkg kricikdnvm iagipmlngs kmlegfvphm 121 datvvsrild eageivaktt cedlcfsggs htsypwpvln prnpeymagg sssgsavava 181 sgycdmavgg dqggsirips swvgiyglkp thglvpytga fsieptldhl gpmantvkdv 241 allleviagr deldsrqpds lppppvkpys klidgdvkdm kvgivkegfn wsnsekdvde 301 lvldsakkle dygikvedts iplhrmgldi wtpiaiegat atmilgsgvg wgrkglfetq 361 iadffgnslk srardlpntv kgvlmlgylm ikmynnryya karnlsivlk eaydsalrky 421 dalimpttpm kamryksepg fdeyfimalg minntapfdv tghpamnipv gysnglpvgl 481 miigrhfeed kvlklanvfe rikk >B: 1 mtvvlptpaq lldvakqcgl sltdadvasf rglmqgsida ynlvgampde lpevkyprtp 61 gyrpspeenp rnawyrkstv kgaasgklkg ktvalkdnim lagvpmmngs atlegyvpdf 121 dativtrmld agaeiagkvh cesfcmsggs htgavgavhn phkmgysagg sssgsgvvva 181 lgevdmaigg dqggsirmps sfcgtygmkp twglvpytgi mpieifvdht gpmtatvadn 241 alllevlagd dgydprikap kveeytkalg qgvkgmkigi lkegfeqpta eaavnesvre 301 aakrfkdlga tvetvsipmh mvgpaiwtpi gtegmtqtmm ygdgyglsrs dlystslmdf 361 hrgwrrqads lsettklfll lgtyinnsfg pryygkalni srrltaaydk afgeydllll 421 pttpmkatkl peptasredy varalemian tapfdithhp aislpcgmvd glpvglmlvg 481 rmfeestiyr aahafeqigd wkkm >C: 1 msiqppdlaa fakaavdgel gikesdlaay haaaegllgs wrtveelyse iapiapkrsw 61 srpaagdngl gawsvqtqlq evssgplagr tvvikdnvsv agvpmmngsr tlegfvpsed 121 atvvtrllga gativgkavc edlcfsggsh tskpgpvrnp wnhahasggs ssgsgalvaa 181 gevdmaiggd qggsiripaa fcgvvghkpt hglvpytggf pieqsidhlg pitrtvtdaa 241 lmlsviagrd ekdprqpdri dpvdyvaalg esakglrvgv vtegfgrens epevddtvrs 301 avetlrsvgl qaadvsipwh lyggalwdvl ategglsqmi dgnaygmnwk ghydpqliey 361 ygrkwrsdps mfsetvklvi lagrhalatq hgrhyamarn laprlaaayd daldqydvlv 421 mptlttrapv laapdgpvee tfsrafemlp qtspfditgh pactvpagla gglpvglmii 481 gkrfhdatvl rvahafenav ggfplpiqer ska >D: 1 mavpppdqka laaaaarygl slspadldsf qpfvhgllas wdaveelysa tapavpqrsw 61 qrpddadnpy nawyvtteir gaadgplagr tvaikdntav agvpmmngsk tvegfvprrd 121 ativtrllaa gatiagkavc edlcfsggsh tsrtgpvrnp wdetrsaggs ssgsaalvas 181 gvvdlatggd qggsvripaa ysgivghkpt hglvpytgaf pieqtidhlg pmtrtvadaa 241 lvldviagpd gldprqpagl vtddysgala rsaaglrvgv vsegfghans dpgvdatvra 301 avdilreagl daedvsvpwh lhgariwdvi ategaaaqmv eangygmnwq geydpeliah 361 ygrqwrarpd efsetvkfvl laggyglase hgrhyamarn lavdlrrayd ealqrydvlv 421 mptlpmtast ipgpdapree iitralemig ntcvtdvtgh patsvpagls gglpvgmmii 481 grmfedatpl rvahtfeqav ggfpaptaat tgsqa >E: 1 mpvrppdene vaalaasygm kptaeevaay tpivaaflts ydaveelyag iapqapddrp 61 strpaeadnp lgawyvttei rgaaegplag rtvavkdnve vagvpmmngs htvegfvpre 121 datvvtrlle agatitgksv cedlcfsggs htsvtgpvrn pwdpsrttgg sssgsaalva 181 sgevdlavgg dqggsirmpa afsgivghkp thglvpytga fpieltldhl gpmtrtvada 241 aamldvlagr dgddprqpad vvpeqyaaal dgdvaglrvg vvaqgfgwee lsedgvddtv 301 raaaerlrel gievvdvdlp whrhalhvwn viatdgatvq mvegngygwn yegrydpeli 361 ahygrqrrev vdrwsptvta valtgrwsle reqtrhyama qnlatevrrq ydaaladvdl 421 lvmptvpmva peipapddpv etrvaralem ivncapfdvs ghpatsvpag lsqglpvgmm 481 lvgrrfedtt plkvahafeq lvggfprppa r >F: 1 mairrpdtqd vaalaesygm hpgedavaty talidgslvs ydaveelyaa iapqapagrs 61 gqrpdddanp lgawyvrtei tgapdgplag rtvavkdnta vagvpmmngs htlegfvptr 121 datvvsrlla agatitgksv cedlcfsggs htsvtgpvrn pwdrsrttgg sssgsgalva 181 agevdlglgg dqggsirmpa afcgvvghkp tfglvpytga fpieltidhl gpitrtvada 241 alaldvlagr dgldprqpad lapeqyaaal ggdvaglrvg vvtqgfgweg lsepgvdetv 301 raaaarlgel gievvevdlp whrhalhvwn viatdgaavq mvegngyghn wdgfydpeli 361 ahygrqsrav adrwsptvtl valagrwsaq qeqtrhyama rdlvvevraq ydraladvdl 421 llmptvpmva plipaaddpl evqvaralem ivntapfdvs ghpatsvpag lsdglpvgmm 481 lvgrrfedtt clkvahafeq ivggfpappt a >G: 1 msvqrpdaea igvaaehfgl hldapareef vglvdgalgs ydvveelyaa svapvtpare 61 yvaeldgksn plgawyvtts vtadapsdtr lagrrvaikd nvalagvpmm ngsrtlegft 121 psrdatvvsr lldagaeiag kavcedlcfs gssftpasgp vrnpwdlsre aggssggsaa 181 lvangdvdla iggdqggsir ipasfcgvvg hkptfglvpy tgafpierti dhvgpiartv 241 adaalmlsvi agydgrdprq pesvsaadyl sslgdgvegm rigvvaegfg heasqaavdd 301 tvqtaaqrft eagavveqvs iqwhrdafhv wnviatdgga yqmldgngyg lnagglydpe 361 qmeyfaehri ahadalsetv klvalcghhg vtalggssyg karnlvplar aaydaalqty 421 dvlvmptlpy vaselpdpsi dratyltkal gmiantapld vtghpalsvp aglvdglpvg 481 mmiigkhhdd atvlrvgqty ehltggfptp ssqragtmgs qfapa >H: 1 mtvirpndda igsaanrygl hldaatrqef aglvdgalgs ydvvddlyad esfsapsrdh 61 atpdasdnpl gawyvttsis tatggpltgr rvaikdnvtv agvpmmngsr slegfvptrd 121 atvvtrllda gatvagkavc edlcfsgssf tpatgpvrnp wdpdreaggs sggsaalvaa 181 gdvdfaiggd qggsiripaa fcgvvghkpt fglvpytgaf piertidhlg pitrtvedaa 241 lllsviagrd ghdprqpdnl vvddyvstld tgtdglrvgv vsqgfghans qpevddavra 301 aahrfteagg vvdevsipwh lhafhvwnvi atdggayqml dgngyglnad glydpelmay 361 faqhriqhad alsetvklva lcghhgvtal ggssygkarn lvpraraayd aalenydvlv 421 mptlpyvase lpaqdvdrvt yvtkalgmia ntapfdvtgh palsvpaglv nglpvgmmvi 481 gkrfddatvl raghafeqir gafptasgqp atsarlsla >I: 1 mpvqrptpeq lrrsaehygl hldadavesf salvdaslls ydaveelhaa qspqpprrqh 61 nkvadgenpl gawyvktdis sdvdgplsgr rvavkdniav agvpmmngsr slegfvpsrd 121 atvvtrllea gatiagkavc edlcfsgash tsatgpvrnp wdptrtsggs ssgsaalvas 181 aevdlaiggd qggsvripss fcgtvghkpt hglvpytgaf pieltidhlg pitktvadaa 241 lmlsvlagtd ghdprqpdgl rpqdyvaald sgvaglrvgv veegfgipgl sdpavdevvr 301 aavarlaeag mevspvsvpw hrhgmhiwnv vategaayqm ldgngyglnw dgsydpelma 361 hygrnrvera dqlsetvklv alcghysltn yqgrhyamar nlanslrqdy dralrsfdvl 421 vmptlpytat plvteqdtre tyveraltmi antapldvsg hpaisvpagl seglpvglml 481 vgkrfddatp lrvahafeql vggfaepprp >J: 1 mpsqtpgvte lspiadaygl glsegelaef vphvsevlrs wdlvdelyer erpdavtrtw 61 aepehnpfga wyvtcdites tdgplagrrv avkdntavag vpmmngsaal egfipdtdat 121 iisrmlaaga tiagkavced lcfsgashts atgpvrnpwd ptrtsggsss gsaalvaagi 181 vdiatggdqg gsvrlpaaft givghkpthg lvpytgafpi eqtldhlgpm trtvadaall 241 lgvlagrdgl dprqptrvav pdyvaelkrp vagmrvgvlc egfghpeavp gvddavrrav 301 dvlraqgmqc envsipwhrh grkiwdviav egattqmvdg naygmnwqga ydpalieffg 361 srrqadgtll pdtvklvllt grhtirrhhg ahyakarnlv prlraaydda lsrfdvlvmp 421 ttplvatpip esdaprdeyl aralemmant agldvsghpa csvpaglvdg lptgmmivgr 481 hfddatvlrv ahayeeatgg flpakvsap >K: 1 mvrvpdpgrl veiarssfgl rlgeeearsf aglmeasias yrrldqlaep slpvkyprtg 61 grrpepeenp lnawyyrcsi kgagegplag krvavkdnva vagvpmmngs atlegyvpef 121 dativtrild aggeisgkav cehlcfsggs htsdtgpvln phdrtrsagg sssgsaalva 181 agevemaigg dqggsirips cwcgtyglkp tyglvpytgv fpieltldhv gpiaasvsdv 241 alllsaiage dgldprqrev rvgdyqnale rgvegvrigv vaegfslpsl seedvdetvr 301 raadtfrelg aevqevsipl hrdgihiwna iaiegatelm vrgngmgtnw eghystslld 361 aywrgrtala gelsetvklt mltgqymrea yggryyarar nlarrlraay daaleradll 421 lmptlpmkat kipapdapre evvaralemi pntcpfdvsg hpamnvpcgt seglpvgmml 481 vgrkwdeetv lraarafekt gayras >L: 1 mpvvpptlee laelaarhgf rlgeadrksf aglveanlaa heaverlytr sapsaphrdy 61 ewpednplga wyvrtriapt gdgplsgrev vikdniavag vpmmngsrav egfvprrdat 121 vvsrvlaagg tivgkavced lcysgssfts asgpvrnpwd parhaggsss gnaallaage 181 adfaiggdqg gsvrmpaafc gvvghkpthg lvpytgafpi ettidhlgpm trtvrdaall 241 lsvlagddgf dprqfrtpep pdylaaveqd irgvrvgvlt egfglpmsep gvddavraaa 301 drlaaagavv sevsvpwhrd gldvwtvigt dgtarqmidg ngygmnvpgr ydpelmvhfa 361 qgrrvhaddm sdtlktmilt gayaaerydm rhyamarela welragydaa ltevdvlvlp 421 tvpyvakplp gpdatreerm inalgmvgnt apfdasghpa isvpaapvdg lptglmiagr 481 rfddamclrv aaayerevgg fpvaar |


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