Jala PCR Kit e tobileng
Nomoro ea katse: HCR2020A
Plant Direct PCR Kit e loketse ho holisa makhasi a semela ka kotloloho, lipeo, joalo-joalo, 'me e ka sebelisoa bakeng sa tlhahlobo e phahameng ea lisampole tsa limela tse se nang li-polysaccharides le polyphenols.DNA polymerase ea amplification e tobileng e itšetlehileng ka ho iphetola ha lintho e na le mamello e phahameng ho feta PCR inhibitors limela.Ho sa le joalo, e boloka ts'ebetso e phahameng ea amplification, e loketseng ho holisa likaroloana tsa DNA ka har'a 5 kb.Sebaka se ikhethileng sa Lysis buffer A ka har'a khiti se ka sebelisoa ho lisa lisele tse ncha kapa tse leqhoa.Ho bonolo ho sebetsa mme lysate e ka sebelisoa e le template ea ho holisa ntle le tlhoekiso.Sistimi ena e na le lisebelisoa tse sireletsang tse etsang hore lisampole tse tala li holisoe hantle ka mor'a ho qhomisoa khafetsa le ho qhibiliha.2 × Plant Direct Master Mix e hloka feela ho eketsa li-primers le li-templates ho etsa amplification reaction, ka hona ho fokotsa ts'ebetso ea liphaephe le ho ntlafatsa ts'ebetso ea ho lemoha le ho hlahisa liphetho.
Likaroloana
| Likaroloana | 50 RXNS | 200 RXNS |
| 2 × Plant Direct Master Mix | 1.25 di ml | 4 × 1.25 di ml |
| Plant Direct Lysis Buffer A | 5 ml | 20 ml |
| Plant Direct Lysis Buffer B* | 5 ml | 20 ml |
*Plant Direct Lysis Buffer B ke sesebelisoa sa boikhethelo, se sebelisetsoang ho fokotsa Plant Direct Lysis Buffer A bakeng sa ho lelefatsa nako ea polokelo ea lisampole.E ka sebelisoa ho latela maemo a sebele.
Maemo a polokelo
2 × Plant Direct Master Mix, boloka ho -30 ~ -15 ℃ 'me u qobe ho qhoqhoa khafetsa le ho qhibiliha;Plant Direct Lysis Buffer, boloka ho -30 ~ -15℃ kapa 2 ~ 8℃.
Mokhoa oa ho Leka
Mohlala oa ho sebetsa-Lekhasi la Semela
Mokhoa o tobileng:Ho khothalletsoa ho sebelisa makhasi a manyenyane.Sebelisa sekoti sa lesoba se nang le bophara bo tsitsitseng ba 0.5 - 3 mm ho fumana sampuli e nyenyane le e ts'oanang, 'me ka ho toba u kenyelle sampuli ho tsamaiso ea PCR (50 μl system e khothalletsoa).Hlokomela, etsa bonnete ba hore sampole e ka tharollo ea PCR eseng khahlano le lebota la tube.Haeba PCR e tobileng e sebelisoa ho matlafatsa likhechana tse telele le lisampole tse rarahaneng, ho sebelisa sampuli e nang le bophara bo fokolang (0.5 - 1 mm) e le template e ka thusa ho fumana liphello tse molemo.
Mokhoa oa ho sila lysis:Ho khothalletsoa ho sebelisa makhasi a manyenyane.Nka lekhasi le lenyane (limilimithara tse ka bang 1 - 3 ka bophara), le behe ho 20 μl Plant Direct Lysis Buffer Ab, 'me u le sile ka hohle kamoo ho ka khonehang (mohato ona o ka etsoa ka ho peta lekhasi ka ntlha ea 100 μl pipette. ho kopanya mohlala).Haeba ho sebelisoa metsoako e meholo ea makhasi (e se ke ea feta 7 mm), eketsa molumo oa buffer ea dilution ho 50 μl.Ka mor'a hore makhasi a be fatše, tharollo e lokela ho bonahala e le tala.Ka mor'a centrifugation e khutšoanyane, eketsa 1 μl ea supernatant ho tsamaiso ea PCR e le templatec reaction.
Mokhoa oa ho futhumatsa mocheso:Ho khothalletsoa ho sebelisa makhasi a manyenyane.Nka karoloana ea lekhasi (e ka bang 1 - 3 mm ka bophara), e behe ka 20 μl Plant Direct Lysis Buffer A, 'me ue chese ka 95 ° C bakeng sa 5 - 10 min.Nako ea lysis e ka atolosoa ka nepo bakeng sa makhasi ao ho leng thata ho a lla (ha ho feta metsotso e 20).Haeba ho sebelisoa metsoako e meholo ea makhasi (e se ke ea feta 7 mm), eketsa molumo oa buffer ea dilution ho 50 μl.Ka mor'a ho futhumatsa, centrifuge hakhutšoanyane, 'me u kenye 1 μl ea supernatant tsamaisong ea PCR e le mokhoa oa ho arabela.
Mohlala oa ho sebetsa–Jala Peo
Mokhoa oa ho sila lysis:Sebelisa scalpel ho seha lipeo ka bophara ba 5 mm, u li kenye ho 100 μl ea Plant Direct Lysis Buffer A, 'me u sile sampole ka ntlha ea pipette kapa lisebelisoa tse ling.Vortex ka bokhutšoanyane 'me u lumelle ho ema mocheso oa kamore bakeng sa 3 - 5 min.Etsa bonnete ba hore sampole ea peō e qoelitsoe ka har'a buffer ea dilution.Kamora nako e khuts'oane ea centrifugation, eketsa 1 μl ea supernatant ho sistimi ea PCR joalo ka template ea karabelo.
Mokhoa oa ho futhumatsa mocheso:Sebelisa scalpel ho seha lipeo ka bophara ba 5 mm, u li kenye ho 100 μl ea Plant Direct Lysis Buffer A, 'me mocheso ho 95 ° C bakeng sa 5 - 10 min.Nako ea lysis e ka atolosoa ka nepo bakeng sa makhasi ao ho leng thata ho a theola (ha a fete metsotso e 30).Ka mor'a ho futhumatsa, centrifuge hakhutšoanyane, 'me u kenye 1 μl supernatant tsamaisong ea PCR e le mokhoa oa ho arabela.
a.Likere kapa lisebelisoa tse ling le tsona li ka sebelisoa ho khaola lisampole tsa boholo bo loketseng;haeba punch kapa lisekere li sebelisoa hape, li lokela ho hloekisoa ka tharollo ea 2% ea sodium hypochlorite pele e sebelisoa ho thibela tšilafalo pakeng tsa lisampole.
b.Etsa bonnete ba hore Plant Direct Lysis Buffer e qhibilihisitsoe ka botlalo pele e sebelisoa.Haeba buffer e le viscous kapa e na le pula, e ka futhumatsoa ho 37 ℃ ho e qhibilihisa ka botlalo pele e sebelisoa.
c.Bophahamo ba thempleite tsamaisong ea karabelo e ka fetoloa ka nepo ho ea ka phapang ea molumo oa thepa ea semela le diluent e ekelitsoeng.
Semela ka ho toba Lysis Buffer
Plant Direct Lysis Buffer A e ka har'a sehlahisoa sena e ntlafalitsoe ka thata ho lokolla mofuta oa lisele tse ngata tsa semela mme e loketse polokelo ea nako e khuts'oane ea limela tse tala ho 4℃.Haeba sampuli e hloka ho bolokoa nako e telele (mohlala, likhoeli tse 1 - 2), ho kgothaletswa ho fetisetsa supernatant ho tube e ncha ea EP le ho e boloka ho -20 ℃.Ho boloka disampole ka tsela e tsitsitseng, eketsa palo e lekanang ya Plant Direct Lysis Buffer B ho supernatant e fetisitsweng, kopanya hantle mme o boloke ho -20 ℃.Nako ea polokelo e tsitsitseng e fapana ho latela mehlala ea limela le linaha.
Reaction System
| ddH2O | Ho isa ho 20.0 μl | Ho isa ho 50.0 μl |
| 2 × Plant Direct Master Mixa | 10.0 µl | 25.0 µ |
| Pele 1 (10 µM) | 0.8µl | 2.0 µl |
| Primer 2 (10 µM)b | 0.8µl | 2.0 µl |
| Mohlala oa lekhasi la semela/motsoako o tala(Sheba ho Sample Processing) | 0.5 - 3 mm lekhasi disc/x µl | 0.5 - 3 mm lekhasi disc/x µl |
a.E na le Mg2+sebakeng sa ho qetela sa 2 mM.
b.Ho kgothaletswa ho sebelisa motsoako oa ho qetela oa 0.4μM bakeng sa primer e 'ngoe le e' ngoe.Tšebeliso e feteletseng ea li-primers e tla lebisa keketseho e sa tobang.
c.Palo ea sampole e sebelisitsoeng e ka fetoloa ho latela maemo a nnete.Chelete e sebelisitsoeng karabelong e le 'ngoe ea sampole ea lysed e tala e ka fetoloa lipakeng tsa 2% - 20% ea kakaretso ea karabelo.Ho sebelisa lisampole tse ngata haholo ho ka baka ho hloleha ha amplification.
Lenaneo la ho arabela
| Mehato | Mocheso | Nako |
| Denaturation ea pele | 98℃ | 5 mets |
| Denaturation | 95℃ | 10 sec |
| Annealing | 58 ~ 72 ℃ | 15 metsotsoana |
| Katoloso | 72℃ | 30 metsotsoana |
| Keketso ea ho Qetela | 72℃ | 5 mets |
a.Denaturation ea Pele (98℃, 5 min) e khothalletsa lysis ea lisele tsa semela, e lokolla DNA ea genomic e ka sebelisoang bakeng sa ho hōlisa PCR.U se ke ua khutsufatsa nako kapa ua fokotsa mocheso.
b.Ho kgothaletswa ho e beha e lekana le boleng ba pele ba Tm kapa 2 ~ 4 ℃ e phahameng ho feta boleng ba Tm.DNA polymerase ea amplification e tobileng e sebelisitsoeng sehlahisoa sena e fapane le e tloaelehileng ea Taq DNA polymerase, 'me e na le litlhoko tse khethehileng bakeng sa mocheso oa ho arabela;Bakeng sa litempele tse rarahaneng, ho holisa ka katleho ho ka finyelloa ka ho lokisa mocheso oa annealing le ho lelefatsa nako ea katoloso.
c.Haeba bolelele ba sehlahisoa sa amplification ke ≤1 kb, nako ea ho eketsa e behiloe ho 30 sec / kb;haeba bolelele ba sehlahisoa sa amplification ke> 1 kb, nako ea katoloso e behiloe ho 60 sec/kb.
d.Bakeng sa lisampole tse rarahaneng kapa lisampole tse nang le chai e tlase ea amplification, palo ea li-cycles e ka eketsoa ka mokhoa o loketseng ho li-cycle tse 40 -50.
Lisebelisoa
E sebetsa bakeng sa ho holisa ka kotloloho lisele tsa semela le tlhahlobo e phahameng ea lisampole tsa semela tse se nang li-polysaccharides le polyphenols.
Lintlha
Bakeng sa tšebeliso ea lipatlisiso feela.Ha e sebelisoe lits'ebetsong tsa tlhahlobo.
1. Bakeng sa ho hōlisa limela tse tala kapa ho hōlisa ka ho toba, ho kgothaletswa ho sebelisa DNA e hloekisitsoeng ea genomic e le taolo e ntle pele o qala teko ho netefatsa hore tsamaiso, li-primers le ts'ebetso li nepahetse.
2. DNA polymerase ea amplification e tobileng e sebelisitsoeng ka har'a khiti ena e na le mosebetsi o matla oa ho hlahloba liphoso.Haeba TA cloning e hloka ho etsoa, ho kgothaletswa ho hloekisa DNA pele o eketsa adenine.
3. Tataiso ea Tlhahiso:
a.Ho khothaletsoa hore setsi sa ho qetela pheletsong ea 3′ e be G kapa C.
b.Ho se lumellane ho latellanang ho lokela ho qojoa litsing tse 8 tsa ho qetela qetellong ea 3′ ea primer.c.Qoba meaho ea hairpin qetellong ea 3' ea primer.
d.Phapang ka boleng ba Tm ea sebatli sa pele le se ka morao ha se ea lokela ho feta 1℃ mme boleng ba Tm bo lokela ho fetoloa ho 60 ~ 72℃ (Primer Premier 5 e khothalletsoa ho bala boleng ba Tm).
e.Tatelano ea tlatsetso ea pele e sa tsamaisaneng le thempleite, ha ea lokela ho kenyelletsoa ha ho baloa boleng ba Tm ea pele.
f.Ho kgothaletswa hore dikahare tsa GC tsa primer e be 40% -60%.
g.Kabo ea kakaretso ea A, G, C le T ho primer e lokela ho ba ka hohle kamoo ho ka khonehang.Qoba ho sebelisa libaka tse nang le litaba tse phahameng tsa GC kapa AT.
h.Qoba boteng ba tatellano e tlatsanang ea li-bases tse 5 kapa ho feta ebang ke ka har'a li-primer kapa lipakeng tsa li-primers tse peli, 'me u qobe ho ba teng ha tatellano e tlatsanang ea li-bases tse 3 kapa ho feta qetellong ea 3′ ea li-primers tse peli.
ke.Sebelisa ts'ebetso ea NCBI BLAST ho lekola boits'oaro ba primer ho thibela ho holisa ka mokhoa o sa tobang.
