2×Rapid Taq Super Mix
Inombolo yekati: HCR2016A
I-2 × i-Rapid Taq Super Mix isekwe kwi-Taq DNA Polymerase elungisiweyo, yongeza into eyomeleleyo yolwandiso, into yokwandisa ukukhulisa kunye nenkqubo ye-buffer ephuculweyo, enobuchule obuphezulu bokukhulisa.Isantya sokukhulisa iitemplates ezintsonkothileyo ezifana negenome ngaphakathi kwe-3 kb ifikelela kwi-1-3 sec/kb, kunye neetemplates ezilula ezifana ne-plasmids ngaphakathi kwe-5 kb ifikelela kwi-1 sec/kb.Le mveliso inokonga kakhulu ixesha lokusabela kwe-PCR.Kwangaxeshanye, umxube uqulathe i-dNTP kunye ne-Mg2+, enokunyuswa kuphela ngokongeza iiprimers kunye neetemplates, ezikwawenza lula kakhulu amanyathelo okusebenza okulinga.Ngapha koko, umxube uqulethe idayi yesalathiso se-electrophoretic, enokuthi ibe yi-electrophoresis ngokuthe ngqo emva kokusabela.I-agent ekhuselayo kule mveliso yenza umxube ugcine umsebenzi ozinzile emva kokuqhwanyaza ngokuphindaphindiweyo kunye nokunyibilika.I-3'-end band A yemveliso ye-PCR inokudityaniswa ngokulula kwi-T vector.
Amacandelo
2×Rapid Taq Super Mix
Iimeko zokuGcina
Iimveliso zePCR Master Mix kufuneka zigcinwe kwi -25~-15 ℃ iminyaka emi-2.
Iinkcukacha
| Ukuchazwa kwemveliso | Rapid Taq Super Mix |
| Ukugxininisa | 2× |
| Isiqalo esishushu | Isiqalo esishushu esakhelwe ngaphakathi |
| I-Overhang | 3′-A |
| Isantya sokusabela | Ngokukhawuleza |
| Ubungakanani (iMveliso yokuGqibela) | Ukuya kuthi ga kwi-15 kb |
| Iimeko zothutho | Umkhenkce owomileyo |
Imiyalelo
1. Inkqubo yokusabela (50 μL)
| Amacandelo | Ubungakanani (μL) |
| Isakhelo se-DNA* | ezifanelekileyo |
| Iprimer yokubhekisa phambili (10 μmol/L) | 2.5 |
| Reverse primer (10 μmol/L) | 2.5 |
| 2×Rapid Taq Super Mix | 25 |
| ddH2O | ukuya ku50 |
2.Ukwandiswa kweProtokholi
| Biyela amanyathelo | Ubushushu (°C) | Ixesha | Imijikelo |
| Ukudalwa kwangaphambili | 94 | 3 imiz | 1 |
| I-Denaturation | 94 | 10 imizuzwana |
28-35 |
| Ukuhlaziya | 60 | 20 imizuzwana | |
| Ulwandiso | 72 | 1-10 imizuzwana/kb |
Ukusetyenziswa okucetyiswayo kweetemplate ezahlukeneyo:
| Uhlobo lwetemplate | Uluhlu losetyenziso lwecandelo (50 μL inkqubo yokusabela) |
| Genomic DNA okanye E. coli ulwelo | 10–1,000 ng |
| I-Plasmid okanye i-viral DNA | 0.5-50 ng |
| cDNA | I-1-5 µL (akukho ngaphezu kwe-1/10 yomthamo opheleleyo we-reaction ye-PCR) |
| Kucetyiswa ukusetyenziswa kweetemplate ezahlukeneyo | |
Amanqaku:
1.Ukusetyenziswa kwe-reagent: yinyibilikisa ngokupheleleyo kwaye udibanise ngaphambi kokusetyenziswa.
2. Ubushushu be-Annealing: Iqondo lokushisa le-annealing lixabiso le-Tm jikelele, kwaye linokusetwa nge-1-2℃ ngaphantsi kwexabiso le-primer Tm.
3. Isantya Sokwandiswa: Setha i-1 sec/kb kwiitemplates ezinzima ezifana ne-genome kunye ne-E. coli ngaphakathi kwe-1 kb;cwangcisa i-3 sec/kb kwiitemplates ezintsonkothileyo ezifana ne-1-3 kb genome kunye ne-E. coli;cwangcisa i-10 sec/kb kwiitemplates ezintsonkothileyo ngaphezulu kwe-3 kb genome kunye ne-E. coli.Ungacwangcisa ixabiso kwi-1 sec/kb kwitemplate elula njenge plasmid engaphantsi kwe 5 kb, 5 sec/kb kwitemplate elula njenge plasmid phakathi kwe 5 kunye 10 kb, kunye ne 10 sec/kb ye template elula. njenge plasmid enkulu kune 10 kb.
Amanqaku
1. Ukhuseleko nempilo yakho, nceda unxibe iidyasi zaselebhu kunye neeglavu ezilahlwayo xa usebenza.
2. Le mveliso isetyenziselwa uphando KUPHELA!
