2×HiF Taq plus Master Mix
Inombolo yekati: HCR2014B
I-HIF Taq kunye noMxube oMkhulu (NgeDayi) sisisombululo esilungele ukusetyenziswa kwe-2 × premixed equlethe i-Plus HIF DNA Polymerase, i-dNTPs, kunye ne-buffer ephuculweyo.Ii-antibodies ezimbini ze-monoclonal kubushushu begumbi obunqanda umsebenzi we-polymerase kunye ne-3′→5′exonuclease umsebenzi zongezwa kumxube oyintloko ngokulula kunye ne-Hot Start PCR.I-extension factor yongezwa kwi-master mix ukuze inike i-enzyme amandla okukhulisa iqhekeza elide, ubude be-amplification bunokufikelela kwi-13 kb, i-enzyme inomsebenzi we-5′→3′ DNA polymerase kunye ne-3′→5′. umsebenzi we-exonuclease, ukuthembeka kwayo ngamaxesha angama-83 e-Taq DNA polymerase, ephindwe ka-9 kwi-polymerase ye-DNA eqhelekileyo.Kufanelekile ukukhulisa iitemplates ezinzima, imveliso yokukhulisa isiphelo esibi.
I-2 × i-HIF Taq kunye noMxube oMkhulu (NgeDayi) ineenzuzo zokukhawuleza nokulula, uvakalelo oluphezulu, ukuchaneka okuqinileyo, uzinzo oluhle, njl. iprotocol yenyathelo, ukwenza lula amanyathelo okulinga kunye nokugcina ixesha.Le mveliso iqulethe i-electrophoresis indicator dyes, kwaye iimveliso ze-PCR zingasetyenziselwa ngokuthe ngqo kwi-electrophoresis.Ukongeza, imveliso ikwaqulethe iarhente ethile ekhuselayo, ukuze umxube wenkosi unokugcina umsebenzi ozinzileyo emva kokunyibilika okuphindaphindiweyo komkhenkce.
Iimeko zokuGcina
Iimveliso kufuneka zigcinwe kwi -25 ~ -15 ℃ unyaka omnye.
Iinkcukacha
| Ukuchazwa kwemveliso | Master Mix |
| Ukugxininisa | 2× |
| Isiqalo esishushu | Isiqalo esishushu esakhelwe ngaphakathi |
| I-Overhang | Buthuntu |
| Isantya sokusabela | Ngokukhawuleza |
| Ubungakanani (iMveliso yokuGqibela) | Ukuya kuthi ga kwi-13kb |
| Iimeko zothutho | Umkhenkce owomileyo |
| Uhlobo lwemveliso | Ukunyaniseka okuphezulu kwePCR premixes |
Imiyalelo
1.PCR Reaction System
| Amacandelo | Umthamo (μL) |
| Isakhelo seDNA | Ifanelekile |
| Iprimer yokubhekisa phambili (10 μmol/L) | 2.5 |
| Reverse Primer (10 μmol/L) | 2.5 |
| 2×HIF Taq kunye noMxube oMkhulu | 25 |
| ddH2O | ukuya ku50 |
2.Kucetyiswa ukusetyenziswa kweetemplate ezahlukeneyo
| Uhlobo lwetemplate | Ukukhulisa amaqhekeza ukusuka kwi-1kb ukuya kwi-10 kb |
| Genomic DNA | 50ng-200 ng |
| I-Plasmid okanye i-Viral DNA | 10pg-20ng |
| cDNA | 1-2.5 µL (Ungagqithi kwi-10% yomthamo wokugqibela we-PCR) |
3.Ukwandiswa kweProtokholi
1) Iprothokholi enamanyathelo amabini (itemplate entsonkothileyo)
| Inyathelo lomjikelo | Temp. | Ixesha | Imijikelo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10sec | 30-35 |
| Ulwandiso | 68℃ | 30 imizuzwana/kb | |
| Ukongezwa kokugqibela | 72℃ | 5 imiz | 1 |
2) Iprothokholi eneNqanaba-ntathu (iprothokholi eqhelekileyo)
| Inyathelo lomjikelo | Temp. | Ixesha | Imijikelo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10sec | 30-35 |
| Ukuhlaziya | 60℃ | 20 imizuzwana | |
| Ulwandiso | 72℃ | 30 imizuzwana/kb | |
| Ukongezwa kokugqibela | 72℃ | 5 imiz | 1 |
3) I-Anealing Gradient Protocol (itemplate entsonkothileyo)
| Inyathelo lomjikelo | Ubushushu | Ixesha | Imijikelo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10 imizuzwana | I-151℃ ukunciphisa ngomjikelo ngamnye) |
| Ukunciphisa igradient | 70-55℃ | 20 imizuzwana | |
| Ulwandiso | 72℃ | 30 imizuzwana/kb | |
| I-Denaturation | 98℃ | 10 imizuzwana |
20 |
| Ukuhlaziya | 55℃ | 20 imizuzwana | |
| Ulwandiso | 72℃ | 30 imizuzwana/kb | |
| Ukongezwa kokugqibela | 72℃ | 5 imiz | 1 |
Iimpawu phantsi kweprotocol yolwandiso olwahlukileyo
| Iprotocol | Amanyathelo amabini | Amanyathelo amathathu | Ukunciphisa igradient |
| Spec. | ngokukhawuleza | phakathi | kade |
| Ukuchaza ngokuthe ngqo | phezulu | phakathi | phezulu |
| PCR isivuno | phakathi | phezulu | phakathi |
| Izinga lokufunyanwa | phezulu | phakathi | phezulu |
Amanqaku
Nceda unxibe iPPE eyimfuneko, idyasi yelebhu kunye neeglavu, ukuqinisekisa impilo kunye nokhuseleko lwakho!
