I-2 × PCR Master Mix (ngaphandle kweDayi)
I-PCR Master Mix luhlobo lwesisombululo esiqhelekileyo se-PCR esilungele ukusetyenziswa, kuquka i-Taq DNA Polymerase, i-dNTP mix MgCl2 kunye ne-buffer ephuculweyo.Ngexesha lokuphendula, kuphela i-primer kunye netemplate inokongezwa ukwenzela ukukhulisa, okwenza lula kakhulu amanyathelo okusebenza okulinga.Le mveliso inezinzisi ezigqwesileyo kwaye inokugcinwa iinyanga ezi-3 kwi-4℃.Imveliso ye-PCR ine-3'-dA protrusion kwaye inokuqulunqwa ngokulula kwi-T vector.
Iimeko zokuGcina
Imveliso kufuneka igcinwe ku -25 ℃~ -15 ℃ iminyaka emibini.
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 (2×) |
Imiyalelo
1.Indlela yokusabela
| Amacandelo | Ubungakanani (μL) |
| Isakhelo seDNA | Ifanelekile |
| Iprimer 1 (10 μmol/L) | 2 |
| I-Primer 2 (10 μmol/L) | 2 |
| PCR Master Mix | 25 |
| ddH2O | Ukuya kwi50 |
2.Ukwandiswa kweProtokholi
| Biyela amanyathelo | Ubushushu (°C) | Ixesha | Imijikelo |
| I-denaturation yangaphambili | 94 ℃ | 5 imiz | 1 |
| I-Denaturation | 94 ℃ | 30 imizuzwana | 35 |
| Ukuhlaziya | 50-60 ℃ | 30 imizuzwana | |
| Ulwandiso | 72 ℃ | 30-60sec/kb | |
| Ukwandiswa kokugqibela | 72 ℃ | 10mins | 1 |
Amanqaku:
1) Ukusetyenziswa kwesifanekiso: 50-200 ng genomic DNA;0.1- 10 ng plasmid DNA.
2) uMg2+ugxininiso: Le mveliso iqulethe i-3 mM ye-MgCl2 efanelekileyo kwiimpendulo ezininzi ze-PCR.
I-3) Iqondo lokushisa le-Anealing: Nceda ubhekisele kwixabiso le-Tm yethiyori 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-cloning yemfuza, 60isekhondi/kb iyacetyiswa.
Amanqaku
1.Ukhuseleko kunye nempilo yakho, nceda unxibe iidyasi zaselebhu kunye neeglavu ezilahlwayo xa usebenza.
2.Isetyenziselwa uphando kuphela!
