2×Rapid Taq Super Mix
Inombolo yekati: HCR2016A
I-2×Rapid Taq Super Mix isekelwe ku-Taq DNA Polymerase eguquliwe, yengeza isici esinamandla sokunweba, isici esithuthukisiwe sokukhulisa i-amplification kanye nesistimu ye-buffer ethuthukisiwe, enekhono eliphezulu lokukhuliswa okuphezulu.Isivinini sokukhulisa izifanekiso eziyinkimbinkimbi ezifana negenome phakathi kuka-3 kb sifinyelela ku-1-3 sec/kb, futhi leso sezifanekiso ezilula njengama-plasmids ngaphakathi kuka-5 kb sifinyelela ku-1 sec/kb.Lo mkhiqizo ungasindisa kakhulu isikhathi sokusabela se-PCR.Ngesikhathi esifanayo, imiksi iqukethe i-dNTP ne-Mg2+, engakhuliswa kuphela ngokungeza ama-primers nezifanekiso, okuphinde kube lula kakhulu izinyathelo zokusebenza zokuhlola.Ngaphezu kwalokho, ukuxuba kuqukethe udayi wenkomba ye-electrophoretic, okungaba yi-electrophoresis ngqo ngemuva kokusabela.I-ejenti evikelayo kulo mkhiqizo yenza ingxube igcine umsebenzi ozinzile ngemva kokuqhwaza okuphindaphindiwe nokuncibilika.Ibhendi engu-3'-end A yomkhiqizo we-PCR ingahlanganiswa kalula ibe yi-T vector.
Izingxenye
2×Rapid Taq Super Mix
Izimo Zokugcina
Imikhiqizo ye-PCR Master Mix kufanele igcinwe ku -25~-15℃ iminyaka emi-2.
Imininingwane
| Ukucaciswa komkhiqizo | I-Rapid Taq Super Mix |
| Ukugxila | 2× |
| Isiqalo Esishisayo | I-Hot Start eyakhelwe ngaphakathi |
| I-Overhang | 3'-A |
| Isivinini sokusabela | Ngokushesha |
| Usayizi (Umkhiqizo Wokugcina) | Kufika ku-15 kb |
| Izimo zokuhamba | Iqhwa elomile |
Iziyalezo
1. Isistimu yokusabela (50 μL)
| Izingxenye | Usayizi (μL) |
| I-DNA yesifanekiso* | ezifanele |
| I-primer eya phambili (10 μmol/L) | 2.5 |
| I-primer ehlehlayo (10 μmol/L) | 2.5 |
| 2×Rapid Taq Super Mix | 25 |
| ddH2O | kuye 50 |
2.I-Amplification Protocol
| Izinyathelo zomjikelezo | Izinga lokushisa (°C) | Isikhathi | Imijikelezo |
| Ukudalwa ngaphambilini | 94 | 3 imiz | 1 |
| I-Denaturation | 94 | 10 isekhondi |
28-35 |
| Anealing | 60 | 20 isekhondi | |
| Isandiso | 72 | 1-10 isekhondi/kb |
Ukusetshenziswa okunconyiwe kwezifanekiso ezahlukahlukene:
| Uhlobo lwesifanekiso | Ibanga lokusetshenziswa kwengxenye (isistimu yokusabela engu-50 μL) |
| I-Genomic DNA noma i-E. coli liquid | 10–1,000 ng |
| I-Plasmid noma i-viral DNA | 0.5-50 ng |
| cDNA | 1-5 µL (akukho ngaphezu kwe-1/10 yevolumu ephelele yokusabela kwe-PCR) |
| Ukusetshenziswa okunconyiwe kwezifanekiso ezahlukahlukene | |
Amanothi:
1.Ukusetshenziswa kwe-reagent: Ncibilikisa ngokuphelele futhi uxube ngaphambi kokusetshenziswa.
2. Izinga lokushisa le-annealing: Izinga lokushisa le-annealing liyivelu ye-Tm yendawo yonke, futhi ingasethwa ngo-1-2℃ ibe ngaphansi kwevelu ye-Tm yokuqala.
3. Isivinini sesandiso: Setha isekhondi elingu-1/kb lezifanekiso eziyinkimbinkimbi njenge-genome ne-E. coli ngaphakathi kuka-1 kb;setha amasekhondi angu-3/kb ukuze uthole izifanekiso eziyinkimbinkimbi njenge-1-3 kb genome kanye ne-E. coli;setha isekhondi elingu-10/kb ukuze uthole izifanekiso eziyinkimbinkimbi ezingaphezu kuka-3 kb genome kanye ne-E. coli.Ungasetha inani libe yisekhondi elingu-1/kb ukuze uthole isifanekiso esilula njenge-plasmid engaphansi kuka-5 kb, 5 sec/kb ukuze uthole isifanekiso esilula njenge-plasmid ephakathi kuka-5 no-10 kb, kanye nesekhondi elingu-10/kb ukuze uthole isifanekiso esilula. njenge-plasmid enkulu kuno-10 kb.
Amanothi
1. Ukuze uphephe kanye nempilo yakho, sicela ugqoke amajazi elebhu namagilavu alahlwayo ukuze usebenze.
2. Lo mkhiqizo owokusetshenziswa ucwaningo KUPHELA!
