Qalabka PCR ee tooska ah ee dhirta
Bisadda No: HCR2020A
Qalabka PCR ee Plant Direct wuxuu ku habboon yahay kor u qaadida tooska ah ee caleemaha dhirta, miraha, iwm., waxaana loo isticmaali karaa baarista sare-u-qaadista ee muunada dhirta ee aan ku jirin polysaccharides iyo polyphenols.Kordhinta tooska ah ee DNA-ga polymerase ee ku salaysan kobaca la toosay ayaa dulqaad sare u leh hor-joogayaasha PCR ee dhirta.Dhanka kale, waxay ilaalisaa waxqabadka sare ee kor u kaca, kaas oo ku habboon kordhinta jajabyada DNA-da gudaha 5 kb.Kaydka Lysis ee gaarka ah ee A ee xirmada ku jira ayaa loo isticmaali karaa in lagu nadiifiyo unugyada dhirta cusub ama barafaysan.Way fududahay in la shaqeeyo oo lysate-ka waxaa loo isticmaali karaa qaab-kordhinta iyada oo aan la nadiifin.Nidaamku wuxuu ka kooban yahay walxo ilaalinaya oo awood u siinaya shaybaarrada cayriin in si wax ku ool ah loo kordhiyo ka dib qaboojinta soo noqnoqda iyo dhalaalidda.2 × Plant Direct Master Mix kaliya waxa uu u baahan yahay in lagu daro aasaasayaal iyo qaabyaal si loo sameeyo falcelin-weyneyn, si loo yareeyo hawlgallada tuubooyinka iyo hagaajinta soo saarista ogaanshaha iyo soo saarista natiijooyinka.
Qaybaha
| Qaybaha | 50 RXNS | 200 RXNS |
| 2 × Dhirta Tooska ah Master Mix | 1.25 ml | 4×1.25 ml |
| Plant Direct Lysis Buffer A | 5 ml | 20 ml |
| Plant Direct Lysis Buffer B* | 5 ml | 20 ml |
* Plant Direct Lysis Buffer B waa reagent ikhtiyaari ah, kaas oo loo isticmaalo in laga takhaluso Plant Direct Lysis Buffer A si loo kordhiyo wakhtiga kaydinta shaybaarada.Waxaa loo isticmaali karaa iyadoo loo eegayo xaaladda dhabta ah.
Xaaladaha Kaydinta
2 × Plant Direct Master Mix, ku kaydi -30 ~ -15℃ oo iska ilaali qaboojinta soo noqnoqda iyo dhalaalidda;Plant Direct Lysis Buffer, ku kaydi -30 ~ -15℃ ama 2 ~ 8℃.
Habka tijaabada
Sambalka Habaynta-Caleenta Dhirta
Habka tooska ah:Waxaa lagu talinayaa in la isticmaalo caleemaha yaryar.Isticmaal feedh dalool leh dhexroor go'an oo ah 0.5 - 3 mm si aad u hesho muunad yar oo labis ah, ka dibna si toos ah muunadda ugu dar nidaamka PCR (Nidaamka 50 μl ayaa lagu talinayaa).Xusuusnow, hubi in muunadku ku jiro xalka PCR oo aan liddi ku ahayn gidaarka tuubada.Haddii PCR tooska ah loo isticmaalo in lagu kordhiyo jajabyo dhaadheer iyo muunado isku dhafan, iyadoo la isticmaalayo muunad leh dhexroor ka yar (0.5 – 1 mm) qaab template ah waxay kaa caawin kartaa inaad hesho natiijooyin wanaagsan.
Habka lysis shiidida:Waxaa lagu talinayaa in la isticmaalo caleemaha yaryar.Qaado cad yar oo caleen ah (qiyaastii 1 - 3 mm dhexroor), ku rid 20 μl Plant Direct Lysis Buffer Ab, oo shiid intii suurtogal ah (tallaabadaan waxaa lagu samayn karaa adigoo ku tuujinaya caleen caarad 100 μl pipette ah. si loo burburiyo saamiga) .Haddii qiyaaso waaweyn oo unugyo caleen ah la isticmaalo (ha ka badnayn 7 mm), kordhi mugga kaydinta qasitaan ilaa 50 μl.Ka dib caleemaha dhulka, xalku waa inuu u muuqdaa cagaar.Ka dib centrifugation kooban, ku dar 1 μl oo ah supernatant nidaamka PCR sida qaab falcelin ah.
Habka lysis kulaylka:Waxaa lagu talinayaa in la isticmaalo caleemaha yaryar.Qaado cad yar oo caleen ah (qiyaastii 1 - 3 mm dhexroor), ku rid 20 μl Plant Direct Lysis Buffer A, oo ku kululee 95 ° C 5 - 10 daqiiqo.Waqtiga lysis si habboon ayaa loo kordhin karaa caleemaha ay adag tahay in la lyse (aan ka badnayn 20 daqiiqo).Haddii qiyaaso waaweyn oo unugyo caleen ah la isticmaalo (ha ka badnayn 7 mm), kordhi mugga kaydinta qasitaan ilaa 50 μl.Kuleyl ka dib, si kooban u qabooji, oo ku dar 1 μl oo ah supernatant nidaamka PCR ahaan qaab falcelin ah.
Sambalka Habaynta–Iniinta dhirta
Habka lysis shiidida:Isticmaal maqaar si aad u gooyso abuurka dhexroorkiisu yahay 5 mm, ku dar 100 μl oo ah Plant Direct Lysis Buffer A, oo ku shiid muunada caarada pipette ama qalab kale.Vortex si kooban oo u ogolow heerkulka qolka 3 - 5 daqiiqo.Hubi in muunada iniinuhu ay ku dhex milmeen kaydiyaha milanka.Ka dib centrifugation kooban, ku dar 1 μl oo ah supernatant nidaamka PCR ahaan qaab falcelin ah.
Habka lysis kulaylka:Isticmaal maqaarka maqaarka si aad u gooyso abuurka dhexroorkiisu yahay 5 mm, ku dar 100 μl ee Plant Direct Lysis Buffer A, kuleylna 95°C 5 – 10 daqiiqo.Waqtiga lysis si habboon ayaa loogu kordhin karaa caleemaha ay adag tahay in la lyse (aan ka badnayn 30 daqiiqo).Kuleyl ka dib, centrifuge si kooban, oo ku dar 1 μl supernatant nidaamka PCR ahaan qaab falcelin ah.
a.Maqasyo ama qalab kale ayaa sidoo kale loo isticmaali karaa in lagu gooyo muunado cabbir ku habboon;Haddii feerka ama maqasyada dib loo isticmaalo, waa in lagu nadiifiyaa 2% sodium hypochlorite ka hor isticmaal kasta si looga hortago wasakhowga u dhexeeya shaybaarada.
b.Hubi in Plant Direct Lysis Buffer uu si buuxda u dhalaalay ka hor isticmaalka.Haddii bakhaarku uu yahay viscous ama uu leeyahay soo degeyaal, waxaa lagu kululayn karaa 37 ℃ si loo dhalaaliyo gebi ahaanba kahor isticmaalka.
c.Mugga template ee habka falcelinta waxaa lagu hagaajin karaa si habboon iyadoo loo eegayo farqiga u dhexeeya mugga alaabta dhirta iyo dareeraha lagu daray.
Plant Direct Lysis Buffer
Plant Direct Lysis Buffer A ee ku jira alaabtan ayaa si adag loo hagaajiyey si loo sii daayo genome ee unugyada dhirta intooda badan oo ku habboon kaydinta muddada gaaban ee dhirta cayriin ee 4 ℃.Haddii shaybaarku u baahan yahay in lagu kaydiyo wakhti dheer (tusaale, 1-2 bilood), waxa lagu talinayaa in lagu wareejiyo tuubada EP cusub oo lagu kaydiyo -20℃.Si aad u kaydiso shaybaarada si adag, ku dar mug siman oo ah Plant Direct Lysis Buffer B ee dufanka la wareejiyay, si fiican u qas oo ku kaydi -20 ℃.Wakhtiga kaydinta xasiloon wuxuu ku kala duwan yahay muunadaha dhirta iyo gobolada.
Nidaamka falcelinta
| ddH2O | Ilaa 20.0 µl | Ilaa 50.0 µl |
| 2 × Dhirta Tooska ah Master Mixa | 10.0 µl | 25.0 µ |
| Hore 1 (10 µM) | 0.8 µl | 2.0 µl |
| Hore 2 (10 µM)b | 0.8 µl | 2.0 µl |
| Muunad caleen/caleeb laga soosaaray(Tixraac Tusaalaha Habaynta) | 0.5 - 3 mm saxan caleen / x µl | 0.5 - 3 mm saxan caleen / x µl |
a.Waxa ku jira Mg2+xadiga ugu dambeeya ee 2mM.
b.Waxaa lagu talinayaa in la isticmaalo fiirsashada kama dambaysta ah ee 0.4μM qalab kasta.Isticmaalka xad-dhaafka ah ee alwaaxyada waxay horseedi doontaa kordhinta kordhinta aan gaarka ahayn.
c.Qadarka muunada la isticmaalay waxaa lagu hagaajin karaa iyadoo loo eegayo xaaladda dhabta ah.Qadarka loo isticmaalo hal fal-celin oo muunad cayriin ah ayaa lagu hagaajin karaa inta u dhaxaysa 2% - 20% wadarta mugga falcelinta.Isticmaalka muunado badan waxay keeni kartaa guul-darreysiga.
Barnaamijka falcelinta
| Talaabooyinka | Heerkulka | Waqtiga |
| Denaturation bilowga ah | 98℃ | 5 daqiiqo |
| Denaturation | 95℃ | 10 ilbiriqsi |
| Annealing | 58 ~ 72℃ | 15 ilbiriqsi |
| Kordhinta | 72℃ | 30 ilbiriqsi |
| Kordhinta u dambaysa | 72℃ | 5 daqiiqo |
a.Denaturation bilowga ah (98 ℃, 5 min) waxay kor u qaadaa lysis ee unugyada dhirta, sii daaya DNA-da genomic oo loo isticmaali karo kordhinta PCR.Ha gaabin wakhtiga hana yareynin heerkulka.
b.Waxaa lagu talinayaa in loo dhigo mid la mid ah qiimaha hore ee Tm ama 2 ~ 4℃ ka sarreeya qiimaha Tm.Kordhinta tooska ah ee DNA polymerase ee loo isticmaalo badeecadan way ka duwan tahay taq DNA-ga polymerase ee caadiga ah, waxayna leedahay shuruudo gaar ah oo loogu talagalay heerkulka falcelinta falcelinta;adeegsiga heerkulka kor u qaadista ayaa si wax ku ool ah u yareeyn karta kor u qaadis aan gaar ahayn waxayna wanaajisaa waxtarka kordhinta.Qaababka kakan, kor u qaadida wax ku oolka ah waxaa lagu gaari karaa iyadoo la hagaajinayo heerkulka dajinta iyo kordhinta waqtiga.
c.Haddii dhererka alaab-weyneyntu uu yahay ≤1 kb, wakhtiga kordhinta waxaa lagu dejiyey 30 sek/kb;haddii dhererka badeecada cod-weynayntu uu yahay>1 kb, wakhtiga kordhinta waxa lagu dejiyay 60 sek/kb.
d.Shaybaarada adag ama shaybaarada leh dhalidda kordhinta yar, tirada wareegyada si habboon ayaa loo kordhin karaa ilaa 40 -50 wareegyo.
Codsiyada
Waxaa lagu dabaqi karaa kor u qaadista tooska ah ee unugyada dhirta iyo baarista sare ee shaybaarada dhirta ee aan ku jirin polysaccharides iyo polyphenols.
Xusuusin
Isticmaalka cilmi-baarista kaliya.Looma isticmaalo hababka ogaanshaha.
1. Kordhinta dhirta cayriin ama kor u qaadida tooska ah, waxaa lagu talinayaa in la isticmaalo DNA-ga la safeeyey si loo xakameeyo kahor intaan la bilaabin tijaabada si loo hubiyo in nidaamka, asaasiga iyo hawlgallada ay sax yihiin.
2. Kordhinta tooska ah ee DNA-ga polymerase ee lagu isticmaalo xirmadan waxay leedahay hawlo akhrin adag.Haddii TA cloning loo baahan yahay in la sameeyo, waxaa lagula talinayaa in la nadiifiyo DNA ka hor inta aan lagu darin adenine.
3. Hagida Naqshadeynta Hore:
a.Waxaa lagu talinayaa in saldhigga ugu dambeeya ee 3′ dhamaadka saleeyaha uu noqdo G ama C.
b.Isku-dheellitir la'aanta isku xigta waa in laga fogaadaa 8-da saldhig ee ugu dambeeya 3′ dhammaadka bilowga.c.Ka fogow qaab dhismeedka timaha biibiile ee 3′ dhamaadka furaha.
d.Farqiga u dhexeeya qiimaha Tm ee hore-u-sameeyaha iyo kaaliyaha dambe waa in aanu ka badnayn 1℃ qiimaha Tmna waa in lagu hagaajiyaa 60 ~ 72℃ (Primer Premier 5 waxa lagu talinayaa in la xisaabiyo qiimaha Tm).
e.Taxanayaal hordhac ah oo dheeraad ah oo aan ku habboonayn qaab-dhismeedka, waa in aan lagu darin marka la xisaabinayo ra'iisul-wasaaraha qiimaha Tm.
f.Waxaa lagu talinayaa in maadada GC ee asaasiga ahi ay noqoto 40% -60%.
g.Qaybinta guud ee A, G, C iyo T ee ku jira furaha waa in ay ahaataa sida ugu macquulsan.Iska ilaali inaad isticmaasho gobollada leh GC ama AT sare.
h.Ka fogow joogitaanka silsilado is-kaabaya oo ah 5 ama in ka badan ha ahaato gudaha furaha ama inta u dhaxaysa laba saldhig oo ka fogow joogitaanka taxane is-kaabaya oo ah 3 ama in ka badan oo ah 3′ dhammaadka labada saldhig.
i.Adeegso shaqada NCBI BLAST si aad u hubiso gaar ahaanshaha furaha si aad uga hortagto kordhinta aan gaarka ahayn.
