Tushen ƙira na farko (ana iya magance matsalolin 99%)
1. Tsawon farko: Littafin koyarwa yana buƙatar 15-30bp, yawanci kusan 20bp.Ainihin yanayin yana da kyau ya zama 18-24bp don tabbatar da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai, amma mafi tsayi mafi kyau, tsayi mai tsayi kuma zai rage ƙayyadaddun ƙayyadaddun, da rage yawan amfanin ƙasa.
2. Girman haɓakawa na farko: 200-500bp ya dace, kuma za'a iya fadada guntu zuwa 10kb a ƙarƙashin takamaiman yanayi.
3. Babban tushe: Abubuwan da ke cikin G + C yakamata su kasance 40-60%, ƙarancin haɓakawar G + C da yawa ba shi da kyau, G + C da yawa yana da sauƙin bayyana ƙungiyoyi marasa takamaiman.ATGC shine mafi kyawun rarraba bazuwar, yana guje wa gungu fiye da 5 purine ko pyrimidine nucleotides.Multi-gc don ƙarshen 5′ da tsaka-tsakin jeri don haɓaka kwanciyar hankali, guje wa GC mai arziki a ƙarshen 3′, babu GC don tushe 3 na ƙarshe, ko babu GC don 3 na tushe na 5 na ƙarshe.
4. Guje wa tsarin na biyu a cikin firamare, da kuma guje wa haɓakawa tsakanin firamare guda biyu, musamman ma haɓakawa a ƙarshen 3, in ba haka ba za a samar da dimer ɗin da ba na musamman ba.
5. Tushen a ƙarshen 3 'ƙare na farko, musamman ma na ƙarshe da tushe, ya kamata a haɗa su sosai don guje wa gazawar PCR saboda sansanonin tashoshi marasa daidaituwa.
6. Masu farawa suna da ko za'a iya ƙarawa tare da wuraren tsagewa masu dacewa, kuma jerin abubuwan da aka haɓaka ya kamata su kasance suna da wuraren da suka dace, wanda ke da amfani sosai don nazarin tsagewa ko cloning kwayoyin halitta.
7. Ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa: masu farawa bai kamata su kasance da wani takamaiman homoloji ba tare da wasu jeri a cikin bayanan jerin abubuwan acid nucleic.
8. Koyi don amfani da software: PP5, Oligo6, DNAstar, Vector NTI, primer3 (Wannan ƙirar kan layi yana aiki mafi kyau).
Abubuwan da ke sama na iya magance aƙalla 99% na matsalolin ƙira na farko.
Sarrafa cikakkun bayanai na zane na farko
1. Tsawon farko
Tsawon madaidaicin matakin shine tushe 18 ~ 30.Gabaɗaya, mafi mahimmancin mahimmancin ƙayyadaddun zafin jiki na annealing na firam shine tsayin firam.Ana zaɓin zafin jiki mai rufewa na firamare gabaɗaya (ƙimar Tm -5℃), wasu kuma suna amfani da ƙimar Tm kai tsaye.Za'a iya amfani da hanyoyin da ke biyowa don ƙididdige yawan zafin jiki na ma'auni.
Lokacin da tsayin firikwensin bai wuce 20bp: [4(G+C)+2(A+T)]-5℃
Lokacin da tsayin firikwensin ya fi 20bp: 62.3 ℃+0.41℃(%GC) -500/tsawon-5℃
Bugu da ƙari, ana iya amfani da software da yawa don ƙididdige yawan zafin jiki, ka'idar lissafin za ta bambanta, don haka wani lokaci ƙididdiga na iya samun ƙaramin gibi.Don inganta halayen PCR, mafi guntu na farko waɗanda ke tabbatar da yanayin zafi na ƙasa da 54 ℃ ana amfani da su don mafi kyawun inganci da ƙayyadaddun ƙayyadaddun bayanai.
Gabaɗaya, ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa ke ƙaruwa ga kowane ƙarin nucleotide, ta yadda mafi ƙarancin tsayin matakin farko don yawancin aikace-aikacen shine 18 nucleotides.Matsakaicin tsayin matakin farko ba shi da mahimmanci sosai, galibi yana da alaƙa da ingancin amsawa.Saboda entropy, mafi tsayin firamare, ƙananan ƙimar da yake ɗaurewa don ɗaure ga DNA ɗin da aka yi niyya don samar da ingantaccen samfuri mai ɗaure biyu don DNA polymerase don ɗaure.
Lokacin amfani da software don ƙirƙira abubuwan ƙira, ana iya ƙayyade tsawon abubuwan ƙima ta hanyar ƙimar TM bi da bi, musamman don abubuwan da ake buƙata na ƙimar ƙimar PCR, TM = 60 ℃ ko haka yakamata a sarrafa su.
2.GC abun ciki
Gabaɗaya, abun ciki na G+C a cikin jeri na farko shine 40% ~ 60%, kuma abun ciki na GC da ƙimar Tm biyu na firamare yakamata a daidaita su.Idan na farko yana da halayen GC ko AT mai tsanani, adadin da ya dace na A, T ko G da wutsiya C za a iya ƙarawa zuwa ƙarshen 5' na farko.
3. Zazzaɓin zafi
Zazzabi mai sanyaya ya kamata ya zama ƙasa da 5℃ fiye da yanayin da ba a kwance ba.Idan adadin tushe na farko yana da ƙananan, za'a iya ƙara yawan zafin jiki na annealing daidai, wanda zai iya ƙara ƙayyadaddun PCR.Idan adadin tushe yana da girma, za'a iya rage yawan zafin jiki na annealing daidai.Bambanci tsakanin zafin jiki na annealing tsakanin nau'i-nau'i na 4 ℃ ~ 6 ℃ ba zai shafi yawan amfanin PCR ba, amma daidaitaccen zafin jiki na annealing na nau'i-nau'i iri ɗaya ne, wanda zai iya bambanta tsakanin 55 ℃ ~ 75 ℃.
4. Guji yankin tsarin na biyu na samfurin haɓakawa
Zai fi kyau a guje wa yankin tsarin na biyu na samfuri lokacin zabar guntu mai ƙarfi.Za'a iya yin tsinkaya da ƙididdige tsayayyen tsari na biyu na guntun manufa ta software na kwamfuta mai dacewa, wanda ke taimakawa wajen zaɓin samfuri.Sakamakon gwaji ya nuna cewa faɗaɗa galibi ba ta yin nasara lokacin da makamashin kyauta (△G) na yankin da za a faɗaɗa bai wuce 58.6lkJ/mol ba.
5. Rashin daidaituwa tare da DNA manufa
Lokacin da ƙararrakin jerin DNA na manufa ya yi girma, firamare na iya ɗaure sassa da yawa na DNA ɗin da aka yi niyya, yana haifar da makada da yawa suna bayyana a sakamakon.A wannan lokacin ya zama dole a yi amfani da gwajin software na BLAST, gidan yanar gizon:http://www.ncbi.nlm.nih.gov/BLAST/.Zaɓi Daidaita jeri biyu (bl2seq).
Manna jeri na farko zuwa yanki na 1 da jerin DNA da aka yi niyya zuwa yanki na 2 yana iya musanya, kuma BLAST yana ƙididdige ma'amala, antisense, da sauran yuwuwar, don haka masu amfani ba sa buƙatar lura ko duka sassan sarƙoƙi na hankali ne.Hakanan zaka iya shigar da lambar GI idan kun san lambar GI na jeri a cikin ma'ajin bayanai, don haka ba lallai ne ku liƙa babban sashe na jerin ba.A ƙarshe, danna Daidaita a 3 don ganin idan na'urar tana da rukunin yanar gizo masu kama da juna a cikin DNA ɗin da aka yi niyya.
6. Tashar farko
Ƙarshen 3' na farko shine inda aka fara haɓakawa, don haka yana da mahimmanci don hana rashin daidaituwa daga farawa a can.Ƙarshen 3 ɗin bai kamata ya wuce 3 a jere G ko C ba, saboda wannan zai sa a yi kuskuren jawo na'urar a cikin jerin abubuwan haɓaka G+C.Ƙarshen 3 ba zai iya samar da wani tsari na biyu ba, sai dai a cikin halayen PCR (AS-PCR) na musamman, ba za a iya daidaita ƙarshen 3' na farko ba.Alal misali, idan an haɓaka yankin da aka haɓaka, 3 'karshen primer bai kamata a ƙare ba a matsayi na uku na codon, saboda matsayi na uku na codon yana da wuyar lalacewa, wanda zai shafi ƙayyadaddun ƙayyadaddun da ingancin haɓakawa.Lokacin amfani da abubuwan haɗawa, koma zuwa teburin amfani da codon, kula da fifikon ilimin halitta, kar a yi amfani da firikwensin haɗawa a ƙarshen 3′, kuma yi amfani da babban taro na firamare (1uM-3uM).
7. Tsarin sakandare na farko
Masu tsarawa da kansu bai kamata su sami jerin abubuwan da suka dace ba, in ba haka ba masu gyara da kansu za su ninka cikin sifofin gashin gashi, kuma wannan tsarin na biyu zai shafi ɗaurin gyare-gyare da samfuri saboda tsangwama.Idan aka yi amfani da hukunce-hukuncen wucin gadi, ci gaba da ginshiƙan madogaran abubuwan da suka dace da kansu bai kamata su wuce 3bp ba.Ya kamata a kasance babu complementarity tsakanin biyu primers, musamman ma complementary zoba na 3 'karshen ya kamata a kauce masa don hana samuwar primer dimers.Gabaɗaya, bai kamata a sami fiye da sansanoni guda 4 a jere ba.
8. Ƙara alamomi ko loci
Ƙarshen 5 yana da ɗan tasiri akan ƙayyadaddun haɓakawa kuma saboda haka ana iya canzawa ba tare da shafar ƙayyadaddun haɓakawa ba.gyaggyarawa na farkon 5 'karshen ya haɗa da: ƙara wurin ƙuntatawa enzyme;Labeled biotin, fluorescence, digoxin, Eu3+, da dai sauransu Gabatar da sunadaran daurin DNA jerin;Gabatar da wuraren maye gurbi, shigar da bacewar jerin maye gurbi da gabatar da jerin masu talla, da sauransu. Ƙarin tushe za su yi tasiri ko žasa da ingancin haɓakawa da haɓaka damar samar da dimer na farko, amma dole ne a yi wasu rangwame don mataki na gaba.Ƙarin jerin abubuwan da ba su wanzu akan jerin abubuwan da aka yi niyya ba, kamar wuraren ƙuntatawa da jerin masu tallatawa, ana iya ƙara su zuwa ƙarshen 5′ na firam ɗin ba tare da shafar takamaiman takamaiman ba.Ba a haɗa waɗannan jeri-jerun a cikin ƙididdige ƙimar ƙimar Tm ba, amma yakamata a gwada don dacewa da tsarin sakandare na ciki.
9. Subclones
Yawancin lokaci, PCR shine cloning na farko kawai, sannan muna buƙatar ƙaddamar da ɓangarorin da aka yi niyya zuwa ɓangarori daban-daban, don haka muna buƙatar ƙira ƙarin tushe don aiki na gaba a cikin matakin PCR.
An taƙaita wasu jerin jeri da aka ƙera don subcloning a ƙasa.
An ƙara wurin ƙuntatawa na endonuclease
Ƙara wuraren ƙuntata enzyme shine hanyar da aka fi amfani da ita don ƙaddamar da samfuran PCR.Gabaɗaya, wurin tsagewar tushe guda shida ne, ban da ƙarshen 5 'karshen rukunin yanar gizon yana buƙatar ƙara sansanonin tsaro 2 ~ 3.Koyaya, adadin tushen kariyar da ake buƙata daban-daban enzymes ya bambanta.Misali, SalⅠ baya buƙatar tushe mai kariya, EcoRⅤ yana buƙatar tushe mai kariya 1, Ba Ⅰ yana buƙatar tushe mai kariya 2, kuma Hind Ⅲ yana buƙatar tushe mai kariya 3.
LIC yana ƙara wutsiya
Cikakken sunan LIC shine cloning-Ligation-Independent cloning, hanyar cloning da Navogen ya ƙirƙira musamman don ɓangaren sa na pET vector.Mai ɗaukar pET da aka shirya ta hanyar LIC yana da madaidaitan tushe guda 12-15 masu mannewa, wanda ya dace da madaidaicin ƙarshen saƙon da aka yi niyya.Don dalilai na haɓakawa, jerin firamare 5′ na guntun da aka saka yakamata ya dace da vector LIC.Ayyukan 3′→5′ na ƙazamin T4 DNA polymerase na iya samar da ƙarshen madauri guda ɗaya akan guntun da aka saka bayan ɗan lokaci kaɗan.Saboda samfurin ba za a iya samuwa ne kawai daga annealing juna na shirye-shiryen sa gutsutsu da vector, wannan hanya ne mai sauri da kuma inganci, kuma shi ne directed cloning.
Directed TA clone ƙara wutsiya
TA cloning ya kasa yin niyya ga guntu zuwa cikin vector, don haka daga baya Invitrogen ya gabatar da wani vector wanda zai iya yin niyya don cloning, wanda ya ƙunshi fitattun tushe guda huɗu GTGGS a ƙarshen ɗaya.Sabili da haka, a cikin ƙirar ƙirar PCR, ya kamata a ƙara ƙarin jerin abubuwan da suka dace, don ɓangarorin na iya zama "daidaitacce".
Idan ba ku da lokaci, za ku iya gwada haɗin kai tsaye, haɗa kwayar halitta tare da vector, wanda shine abin da muke kira ET gene synthesis a cikin malaman musecularists.
D. In-Fusion cloning Hanyar
Babu ligase da ake buƙata, babu dogon dauki da ake buƙata.Muddin an gabatar da jeri a ƙarshen ƙarshen madaidaicin layi A cikin ƙirar ƙirar ƙira, to ana ƙara samfurin PCR da vector mai layi a cikin maganin in-fusion enzyme wanda ke dauke da BSA kuma an sanya shi a cikin dakin zafin jiki na rabin sa'a, ana iya yin canji.Wannan hanya ta dace musamman don babban juzu'in juzu'i.
10. Haɗa firamare
Wani lokaci, taƙaitaccen bayanin jeri ne kawai aka sani game da ƙira na farko.Misali, idan jerin amino acid kadai aka san, ana iya ƙera firam ɗin haɗakarwa.Matsakaicin haɗe-haɗe shine cakuda jeri daban-daban wanda ke wakiltar dukkan yuwuwar tushe daban-daban waɗanda ke ɓoye amino acid guda ɗaya.Don haɓaka ƙayyadaddun ƙayyadaddun bayanai, zaku iya komawa zuwa teburin amfani da codon don rage haɗawa bisa ga zaɓin amfani da tushe na ƙwayoyin halitta daban-daban.Ana iya haɗa Hypoxanthine tare da duk sansanonin don rage yawan zafin jiki na fidda.Kar a yi amfani da sansanonin da aka haɗa a ƙarshen 3′ na firamare saboda ɓarkewar tushe 3 na ƙarshe a ƙarshen 3′ ya isa ya fara PCR a wurin da bai dace ba.Ana amfani da mafi girma na firamare (1μM zuwa 3μM) saboda firamare a cikin gaurayawan haɗawa da yawa ba su keɓance ga samfurin manufa ba.
PCR albarkatun kasasarrafawa
1. Babban adadin
Matsakaicin kowane firam shine 0.1 ~ 1umol ko 10 ~ 100pmol.Zai fi kyau don samar da sakamakon da ake buƙata tare da mafi ƙanƙanci na firamare.Babban maida hankali na firamare zai haifar da rashin daidaituwa da haɓakawa mara kyau, kuma yana ƙara damar ƙirƙirar dimers tsakanin firam.
2. Babban maida hankali
Ƙaddamar da ƙaddamarwa yana rinjayar takamaiman.Matsakaicin matsakaicin matakin farko shine gabaɗaya tsakanin 0.1 da 0.5μM.Maɗaukaki mafi girma yana haifar da haɓaka samfuran da ba takamaiman ba.
3. Annealing zafin jiki na farko
Wani muhimmin ma'auni don masu farawa shine zafin jiki na narkewa (Tm).Wannan shine zafin jiki lokacin da kashi 50% na firamare da jeri-nauyi ke wakilta a matsayin kwayoyin halittar DNA guda biyu.Ana buƙatar Tm don saita zafin jiki na PCR.Mahimmanci, zafin zafin jiki na annealing yana da ƙarancin isa don tabbatar da ingantacciyar annealing na firamare tare da jerin maƙasudi, amma yana da girman isa don rage ɗaurin da ba na musamman ba.Madaidaicin zafin jiki na annealing daga 55 ℃ zuwa 70 ℃.Gabaɗaya ana saita zafin zafin jiki na 5℃ ƙasa da Tm na fari.
Akwai dabaru da yawa don saita Tm, waɗanda suka bambanta sosai dangane da dabarar da aka yi amfani da su da kuma jerin abubuwan farko.Saboda mafi yawan ƙididdiga suna ba da ƙimayar ƙimar Tm, duk yanayin zafi mai ɓarna shine kawai mafari.Ana iya inganta ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa ta hanyar nazarin halayen halayen da yawa waɗanda ke haɓaka yanayin zafi a hankali.Fara ƙasa da kimanta Tm-5 ℃, kuma a hankali ƙara yawan zafin jiki a cikin haɓaka na 2℃.Maɗaukakin zafin jiki mai zafi zai rage samuwar dimers na farko da samfuran da ba na musamman ba.Don samun sakamako mafi kyau, abubuwan farko guda biyu ya kamata su kasance da ƙimar ƙimar Tm.Idan bambancin Tm na nau'i-nau'i na farko ya fi 5 ℃, masu farawa za su nuna gagarumin farawar ƙarya ta amfani da ƙananan zafin jiki a cikin sake zagayowar.Idan matakan Tm guda biyu sun bambanta, saita zafin zafi zuwa 5 ℃ ƙasa da mafi ƙarancin Tm.A madadin, don ƙara ƙayyadaddun ƙayyadaddun bayanai, za a iya yin zagayawa guda biyar a farkon yanayin zafi da aka tsara don mafi girma Tm, sannan sauran hawan keken a yanayin zafi da aka tsara don ƙananan Tm.Wannan yana ba da damar samun kwafin samfuri na ƙayyadaddun ƙayyadaddun ƙayyadaddun yanayi.
4. Farko mai tsabta da kwanciyar hankali
Daidaitaccen tsaftar madaidaicin al'ada ya isa ga yawancin aikace-aikacen PCR.Cire ƙungiyoyin benzoyl da isobutylyl ta hanyar desalting kadan ne saboda haka baya tsoma baki tare da PCR.Wasu aikace-aikacen suna buƙatar tsarkakewa don cire duk wani jerin marasa cikakken tsayi a cikin tsarin hadawa.Wadannan jerin sassan da aka yanke suna faruwa ne saboda ingancin haɗin sunadarai na DNA ba 100%.Wannan tsari ne na madauwari wanda ke amfani da maimaita halayen sinadarai yayin da aka ƙara kowane tushe don yin DNA daga 3' zuwa 5'.Kuna iya kasawa a kowane zagaye.Dogayen firamare, musamman waɗanda suka fi ginshiƙai 50, suna da ɗimbin kaso mai yawa na tarkace kuma suna iya buƙatar tsarkakewa.
Abubuwan da ake amfani da su na farko suna shafar ingancin sinadarai na roba da hanyar tsarkakewa.Kamfanonin Biopharmaceutical, irin su Cytology da Shengong, duk suna amfani da ƙaramin OD naúrar don tabbatar da jimillar fitarwa na oligonucleoside.Ana aikawa da maɓalli na musamman a cikin busasshiyar foda.Zai fi kyau a sake narkar da abubuwan da aka gyara a cikin TE domin ƙaddamarwar ƙarshe shine 100μM.TE ya fi ruwan da aka lalata saboda pH na ruwa sau da yawa acidic kuma zai haifar da hydrolysis na oligonucleosides.
Zaman lafiyar firamare ya dogara da yanayin ajiya.Ya kamata a adana busassun foda da narkar da magunguna a -20 ℃.Abubuwan da aka narkar da su a cikin TE a mafi girma fiye da 10μM za a iya adana su a tsaye a -20 ℃ na tsawon watanni 6, amma ana iya adana su kawai a cikin zafin jiki (15 ℃ zuwa 30 ℃) na kasa da mako 1.Ana iya adana busassun busassun foda a -20 C na akalla shekara 1 kuma a cikin zafin jiki (15 C zuwa 30 C) har zuwa watanni 2.
5. Enzymes da yawansu
A halin yanzu, Taq DNA polymerase da aka yi amfani da shi shine ainihin injiniyan injiniyan kwayoyin halitta wanda kwayoyin coliform suka hada.Adadin enzyme da ake buƙata don haɓaka halayen PCR na yau da kullun shine kusan 2.5U (yana nufin jimlar amsawar 100ul).Idan maida hankali ya yi yawa, zai iya haifar da haɓakar da ba ta dace ba;idan maida hankali ya yi ƙasa da ƙasa, za a rage adadin samfuran roba.
6. Quality da maida hankali na dNTP
Ingancin dNTP yana da alaƙa da kusanci da haɓakawa da ingancin haɓaka PCR.DNTP foda yana da ƙima, kuma bambancinsa yana rasa aikin ilimin halitta idan an adana shi ba daidai ba.dNTP bayani yana da acidic, kuma ya kamata a yi amfani da shi a cikin babban taro, tare da 1M NaOH ko 1M Tris.HCL buffer bayani don daidaita PH zuwa 7.0 ~ 7.5, ƙananan ƙananan marufi, daskararre ajiya a -20 ℃.Yawan daskarewa-narkewa zai lalata dNTP.A cikin amsawar PCR, dNTP yakamata ya zama 50 ~ 200umol/L.Musamman, ya kamata a biya hankali ga maida hankali na DNTPS guda huɗu ya zama daidai (daidaitaccen shiri na tawadar Allah).Idan natsuwar ɗayansu ya bambanta da sauran (mafi girma ko ƙasa), za a haifar da rashin daidaituwa.Ƙananan maida hankali zai rage yawan amfanin PCR.dNTP na iya haɗawa tare da Mg2+ kuma ya rage ƙaddamar da Mg2+ kyauta.
7. Samfurin (jinin manufa) nucleic acid
Adadin da matakin tsarkakewa na samfur nucleic acid shine ɗayan mahimman hanyoyin haɗin kai don nasara ko gazawar PCR.Hanyoyin tsarkakewa na DNA na gargajiya yawanci suna amfani da SDS da protease K don narkewa da zubar da samfurori.Babban ayyukan SDS sune: narkar da lipids da sunadaran a jikin kwayar halitta, don haka lalata membrane tantanin halitta ta hanyar narkar da sunadarai na membrane, da rarraba sunadaran nukiliya a cikin tantanin halitta, SDS kuma na iya haɗuwa da sunadaran da haɓaka;Protease K na iya yin hydrolyze da narkar da sunadaran, musamman histones da ke daure da DNA, sannan a yi amfani da phenol mai narkewa da chloroform don fitar da sunadarai da sauran abubuwan da ke cikin tantanin halitta, kuma suyi amfani da ethanol ko isopropyl barasa don haɓaka acid nucleic.Ana iya amfani da acid nucleic da aka fitar azaman samfuri don halayen PCR.Don samfuran ganowa na asibiti gabaɗaya, ana iya amfani da hanya mai sauri da sauƙi don narkar da sel, ƙwayoyin cuta na lysate, narke da cire sunadaran daga chromosomes zuwa ƙwayoyin halitta masu niyya kyauta, kuma ana amfani da su kai tsaye don haɓaka PCR.Hakar samfurin RNA yawanci yana amfani da guanidine isothiocyanate ko hanyar protease K don hana RNase daga wulakanta RNA.
8.Mg2+ maida hankali
Mg2+ yana da tasiri mai mahimmanci akan ƙayyadaddun ƙayyadaddun da yawan amfanin PCR.Gabaɗaya halayen PCR, lokacin da maida hankali na dNTP daban-daban shine 200umol/L, ƙimar da ta dace na Mg2+ shine 1.5 ~ 2.0mmol/L.Matsakaicin Mg2+ ya yi yawa, ƙayyadaddun halayen halayen suna raguwa, haɓakawa mara ƙayyadaddun yana faruwa, ƙarancin maida hankali zai rage ayyukan Taq DNA polymerase, yana haifar da raguwar samfuran amsawa.
Magnesium ions yana shafar bangarori da yawa na PCR, kamar aikin DNA polymerase, wanda ke rinjayar yawan amfanin ƙasa;Wani misali kuma shine ƙaddamarwa na farko, wanda ke shafar takamaiman.dNTP da samfuri suna ɗaure zuwa magnesium ion, rage yawan adadin magnesium ion kyauta da ake buƙata don aikin enzyme.Mafi kyawun maida hankali na ion magnesium ya bambanta don nau'i-nau'i na farko da samfura daban-daban, amma PCR na yau da kullun farawa tare da 200μM dNTP shine 1.5mM (bayanin kula: Don PCR mai ƙididdigewa na ainihi, yi amfani da 3 zuwa 5mM magnesium ion bayani tare da bincike mai kyalli).Maɗaukakin maɗaukaki na ions na magnesium kyauta yana haɓaka yawan amfanin ƙasa, amma kuma yana haɓaka haɓakawa mara iyaka da rage aminci.Don ƙayyade mafi kyawun maida hankali, an yi titration na magnesium ion a cikin haɓakar 0.5mM daga 1mM zuwa 3mM.Don rage dogaro akan inganta ion magnesium, ana iya amfani da Platinum Taq DNA polymerase.Platinum Taq DNA polymerase yana da ikon kiyaye aiki a kan kewayon ma'aunin ma'aunin ion magnesium fiye da Taq DNA polymerase don haka yana buƙatar ƙarancin haɓakawa.
9. Abubuwan haɓakawa na PCr
Haɓaka yanayin zafi mai ɓarna, ƙira na farko, da tattarawar magnesium ion ya wadatar don ƙayyadaddun ƙayyadaddun ƙayyadaddun mafi yawan samfuran;duk da haka, wasu samfuran, gami da waɗanda ke da babban abun ciki na GC, suna buƙatar ƙarin matakan.Abubuwan da ke da alaƙa da yanayin narkewar DNA suna ba da wata hanya don inganta ƙayyadaddun samfur da yawan amfanin ƙasa.Ana buƙatar cikakken denaturation na samfuri don kyakkyawan sakamako.
Bugu da ƙari, tsarin na biyu yana hana ƙaddamarwa na farko da haɓakar enzyme.
Abubuwan ƙari na PCR, gami da foramide, DMSO, glycerin, betaine, da PCRx Enhancer Solution, haɓaka haɓakawa.Hanyar da za su iya amfani da ita ita ce rage yawan zafin jiki na narkewa, don haka taimakawa annealing na kayan aiki da kuma taimakawa wajen fadada DNA polymerase ta hanyar yanki na biyu.Maganin PCRx yana da wasu fa'idodi.Ana buƙatar ƙaramar haɓaka ion magnesium lokacin amfani da Platinum Taq DNA polymerase da Platinum Pfx DNA polymerase.Don haka, ana haɗa fasahar Platinum tare da ƙari don haɓaka ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun rage dogaro ga tsarin na uku, haɓakar magnesium ion.Don sakamako mafi kyau, ya kamata a inganta ƙaddamar da abubuwan ƙari, musamman DMSO, formamide, da glycerol, waɗanda ke hana Taq DNA polymerase.
10. Zafafan farawa
PCR mai zafi yana ɗaya daga cikin mahimman hanyoyin don haɓaka ƙayyadaddun PCR ban da ƙira mai kyau.Kodayake mafi kyawun zafin jiki na Taq DNA polymerase shine 72 ℃, polymerase yana ci gaba da aiki a zafin jiki.Don haka, ana samar da samfurori marasa ƙayyadaddun lokacin da yawan zafin jiki ya kasance ƙasa da zafin jiki na annealing yayin shirye-shiryen amsawar PCR da kuma farkon zagayowar thermal.Da zarar an ƙirƙira, waɗannan samfuran marasa ƙayyadaddun ana haɓaka su yadda ya kamata.PCR mai zafi yana da tasiri musamman lokacin da rukunin yanar gizon da aka yi amfani da su don ƙirƙira na farko sun iyakance ta wurin wuraren abubuwan halitta, kamar maye gurbi da aka jagoranta, cloning magana, ko gini da sarrafa abubuwan kwayoyin halitta da ake amfani da su don injiniyan DNA.
Hanyar gama gari don iyakance ayyukan Taq DNA polymerase shine shirya maganin amsawar PCR akan kankara kuma sanya shi a cikin na'urar PCR da aka rigaya.Wannan hanya mai sauƙi ne kuma maras tsada, amma baya kammala aikin enzyme kuma saboda haka baya kawar da haɓakar samfuran da ba na musamman ba.
Thermal priming yana jinkirta haɗin DNA ta hanyar hana wani muhimmin sashi har sai na'urar PCR ta kai zafin ƙima.Yawancin hanyoyin ƙaddamar da zafin jiki na hannu, gami da jinkirin ƙari na Taq DNA polymerase, suna da wahala, musamman don aikace-aikacen da ake samarwa.Sauran hanyoyin fidda zafin zafi suna amfani da garkuwar kakin zuma don ƙulla wani muhimmin sashi, gami da ions na magnesium ko enzymes, ko don ware abubuwan da suke amsawa ta jiki, kamar samfuri da buffers.Lokacin zagayowar thermal, ana fitar da sassa daban-daban kuma ana haɗa su tare yayin da kakin zuma ya narke.Kamar hanyar farawa mai zafi na hannu, hanyar garkuwar kakin zuma tana da wahala kuma tana da saurin kamuwa da cuta kuma bai dace da aikace-aikacen kayan aiki masu yawa ba.
Platinum DNA polymerase ya dace da inganci don PCR mai zafi ta atomatik.Platinum Taq DNA polymerase ya ƙunshi recombinant Taq DNA polymerase hade da monoclonal antibody da Taq DNA polymerase.PCR ne ke ƙirƙira ƙwayoyin rigakafin don hana ayyukan enzyme yayin riƙe da zafin jiki na tsawon lokaci.An saki Taq DNA polymerase a cikin martani yayin 94 ℃ rufi na matakin denaturation, maido da cikakken aikin polymerase.Ya bambanta da gyare-gyaren sinadarai Taq DNA polymerase don farawa na thermal, Platinum enzyme baya buƙatar dogon rufewa a 94 ℃ (minti 10 zuwa 15) don kunna polymerase.Tare da PlatinumTaq DNA polymerase, 90% na ayyukan Taq DNA polymerase an dawo dasu bayan mintuna 2 a 94 ℃.
11. Nest-PCR
Nasarar ƙarawa ta yin amfani da firam ɗin gida na iya inganta ƙayyadaddun bayanai da azanci.Zagaye na farko shine daidaitaccen haɓakawa na 15 zuwa 20.An narkar da ɗan ƙaramin juzu'in samfurin haɓakawa na farko sau 100 zuwa 1000 kuma an ƙara shi zuwa zagaye na biyu na haɓakawa don zagayowar 15 zuwa 20.A madadin, farkon haɓaka samfurin na iya girma ta hanyar tsarkakewa gel.Ana amfani da firamare na gida a zagaye na biyu na haɓakawa, wanda zai iya ɗaure zuwa jerin maƙasudi a cikin na farko.Amfani da PCR da aka gina yana rage yuwuwar haɓaka wuraren da aka yi niyya da yawa saboda akwai ƴan jeri-jeri da suka dace da saiti biyu na firamare.Jimlar adadin zagayowar (30 zuwa 40) tare da firamare iri ɗaya sun haɓaka wuraren da ba takamaiman ba.PCR Nsted yana ƙara azanci na iyakantaccen jeri na manufa (misali, mrnas ba kasafai) kuma yana haɓaka ƙayyadaddun PCRS masu wahala (misali 5′ RACE).
12. Saukowa PCR
Saukowa PCR yana haɓaka ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai ta hanyar amfani da matsananciyar yanayi don ƴan hawan keke na farko na PCR.Zagayen zagayowar yana farawa ne a yanayin zafi mai raɗaɗi kusan 5 ℃ sama da kiyasin Tm, sannan kowane zagayowar yana raguwa da 1 ℃ zuwa 2 ℃ har sai zafin zafin da ke ƙasa ya kasance ƙasa Tm 5℃.Samfurin makoma kawai tare da mafi girman homology ne za a haɓaka.Waɗannan samfuran suna ci gaba da faɗaɗa cikin zagayowar da ke gaba, suna cunkoso samfuran da ba na musamman ba.Saukowa PCR yana da amfani ga hanyoyin da ba a san matakin homology tsakanin firamare da samfurin manufa ba, kamar AFLP DNA printing.
Abubuwan PCR masu alaƙa
2 × PCR HeroTMTsarin Mix yana da mafi girman juriya ga masu hana PCR fiye da tsarin PCR na yau da kullun, kuma yana iya jurewa cikin sauƙi tare da haɓaka PCR na samfuran hadaddun daban-daban.Tsarin amsawa na musamman da ingantaccen Taq Hero yana sa amsawar PCR ta sami ingantaccen haɓakawa, ƙayyadaddun ƙayyadaddun hankali da hankali.
Ingantacciyar haɓakawa mafi girma
Yana da 5'→3' DNA polymerase aiki da 5'→ 3' ayyukan exonuclease, ba tare da 3'→5' aikin exonuclease ba.
Real Time PCR Easyᵀᴹ-SYBR Green I Kit
Takamaiman - ingantaccen buffer da zafi-farawa Taq enzyme na iya hana haɓaka da ba takamaiman takamaiman ba da samuwar dimer na farko.
Babban hankali-zai iya gano ƙananan kwafin samfuri
Kit ɗin yana amfani da na musamman na Foregene reverse reagent reagent da Foregene HotStar Taq DNA Polymerase haɗe tare da keɓaɓɓen tsarin amsawa don inganta ingantaccen haɓakawa da ƙayyadaddun halayen.
Lokacin aikawa: Mayu-09-2023
