2×PCR Super Mix (ngedayi)
I-2 × PCR Master Mix iqulethe i-Taq DNA Polymerase, i-dNTPs, kunye nezinye izinto ezifunekayo ze-PCR.I-Master Mix izinzile kwiinyanga ezi-3 kwi-4 ℃ kunye nezilungisi zethu ezilungiselelwe.Isisombululo sangaphambili sokuxuba silungiselelwe i-PCR eqhelekileyo kwaye ilungele ukusetyenziswa ngokufaka i-template ye-DNA kunye ne-primers.Iimveliso ze-PCR zinokulayishwa ngokuthe ngqo kwi-electrophoresis kunye nedayi eluhlaza ye-bromophenol elayishiwe ngaphambili.Iimveliso ezandisiweyo ziqulathe i-3 '-dA protrusion kwaye zinokudityaniswa ngokulula zibe yi-T vector.I-2 × PCR Master Mix yenza lula inkqubo ye-PCR kwaye inciphisa ukungcoliseka.
Iimeko zokuGcina
Iimveliso kufuneka zigcinwe ku -25℃~-15℃ iminyaka emi-2.
Iinkcukacha
| Ukunyaniseka(vs.Taq) | 1× |
| Isiqalo esishushu | No |
| I-Overhang | 3 '-A |
| Ipolymerase | Taq DNA Polymerase |
| Ifomathi yokusabela | SuperMix okanye Master Mix |
| Isantya sokuphendula | Umgangatho |
| Uhlobo lweMveliso | PCR Master Mix (2x) |
Imiyalelo
1.Indlela yokusabela
| Amacandelo | Umthamo(μL) |
| Isakhelo seDNA | Ifanelekile |
| Iprimer 1 (10 μmol/L) | 2 |
| I-Primer 2 (10 μmol/L) | 2 |
| I-2 × PCR Master Mix | 25 |
| ddH2O | ukuya ku50 |
2.Ukwandiswa kweProtokholi
| Biyela amanyathelo | Ubushushu (°C) | Ixesha | Imijikelo |
| I-denaturation yokuqala | 94 | 5 imiz | 1 |
| I-Denaturation | 94 | 30 imizuzwana | 35 |
| Ukuhlaziya | 50-60 | 30 imizuzwana | |
| Ulwandiso | 72 | 30-60 imizuzwana/kb | |
| Ukongezwa kokugqibela | 72 | 10 imiz | 1 |
Phawula:
1) Ukusetyenziswa kwesifanekiso: 50-200ng DNA genomic;I-0.1-10ng ye-plasmid DNA.
2) uMg2+ukugxininiswa: Le mveliso iqulethe i-3mM ye-MgCl2, ifanelekile kwiimpendulo ezininzi ze-PCR.
3) Iqondo lobushushu le-Anealing: Nceda ubhekisele kwixabiso lethiyori ye-Tm yeeprimers.Ubushushu be-annealing bunokusetwa kwi-2-5℃ ngaphantsi kwexabiso lethiyori ye-primer.
I-4) Ixesha lokwandiswa: Ukuchonga i-molecular, i-30 sec / kb iyacetyiswa.Kwi-gene cloning, i-60 sec/kb iyacetyiswa.
Amanqaku
1.Iimveliso ze-PCR ezine-2 × PCR Master Mix azifanelekanga kwi-polyacrylamide gel electrophoresis.
2.Ukhuseleko kunye nempilo yakho, nceda unxibe iidyasi zaselebhu kunye neeglavu ezilahlwayo xa usebenza.
3.Isetyenziselwa uphando KUPHELA!
