2×HiF Taq plus Master Mix
Inombolo yekati: HCR2014B
I-HIF Taq plus Master Mix (Ngodayi) iyisixazululo esilungele ukusetshenziswa esingu-2×premixed esiqukethe i-Plus HIF DNA Polymerase, i-dNTPs, kanye nebhafa elungiselelwe.Amasosha omzimba amabili e-monoclonal ezingeni lokushisa legumbi avimbela umsebenzi we-polymerase kanye nomsebenzi we-3′→5′exonuclease engezwa kungxube eyinhloko ukuze kube lula futhi kucace kakhulu i-Hot Start PCR.I-extension factor yengezwa ekuxubeni okuyinhloko ukunikeza i-enzyme amandla okukhulisa izingcezu ezinde, ubude bokukhulisa bungafika ku-13 kb, i-enzyme inomsebenzi we-5′→3′ DNA polymerase kanye no-3′→5′. umsebenzi we-exonuclease, ukwethembeka kwayo kuphindwe izikhathi ezingama-83 kune-Taq DNA polymerase, okuyizikhathi eziyi-9 kune-DNA polymerase evamile.Ilungele ukukhulisa izifanekiso eziyinkimbinkimbi, umkhiqizo wokukhulisa uyisiphetho esibi.
I-2×HIF Taq plus Master Mix(Nge-Dye) inezinzuzo zokushesha nokulula, ukuzwela okuphezulu, ukucaciswa okuqinile, ukuzinza okuhle, njll., uhlelo lokusabela ludinga kuphela ukwengeza iziqalo nezifanekiso, futhi lungakhuliswa nge-two- isinyathelo esilandelwayo, ukwenza lula izinyathelo zokuhlola kanye nokulondoloza isikhathi.Lo mkhiqizo uqukethe odayi bezinkomba ze-electrophoresis, futhi imikhiqizo ye-PCR ingasetshenziswa ngokuqondile ku-electrophoresis.Ngaphezu kwalokho, umkhiqizo uqukethe ne-ejenti ethile evikelayo, ukuze imiksi eyinhloko ikwazi ukugcina umsebenzi ozinzile ngemva kokuncibilika kweqhwa okuphindaphindiwe.
Izimo Zokugcina
Imikhiqizo kufanele igcinwe ku -25~-15℃ unyaka ongu-1.
Imininingwane
| Ukucaciswa komkhiqizo | I-Master Mix |
| Ukugxila | 2× |
| Isiqalo Esishisayo | I-Hot Start eyakhelwe ngaphakathi |
| I-Overhang | Buthuntu |
| Isivinini sokusabela | Ngokushesha |
| Usayizi (Umkhiqizo Wokugcina) | Kufika ku-13kb |
| Izimo zokuhamba | Iqhwa elomile |
| Uhlobo lomkhiqizo | High fidelity PCR premixes |
Iziyalezo
1.I-PCR Reaction System
| Izingxenye | Ivolumu (μL) |
| Isifanekiso se-DNA | Kuyafaneleka |
| I-primer eya phambili (10 μmol/L) | 2.5 |
| I-Primer Reverse (10 μmol/L) | 2.5 |
| 2×HIF Taq plus Master Mix | 25 |
| ddH2O | kuye 50 |
2.Ukusetshenziswa okunconyiwe kwezifanekiso ezahlukahlukene
| Uhlobo lwesifanekiso | Khulisa izingcezu zisuka ku-1kb ziye ku-10kb |
| I-Genomic DNA | 50ng-200 ng |
| I-Plasmid noma i-Viral DNA | 10pg-20ng |
| cDNA | 1-2.5 µL (Ungadluli u-10% wevolumu yokugcina ye-PCR) |
3.I-Amplification Protocol
1) Iphrothokholi Yezinyathelo Ezimbili (isifanekiso esiyinkimbinkimbi)
| Isinyathelo somjikelezo | Temp. | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10isekhondi | 30-35 |
| Isandiso | 68℃ | 30 isekhondi/kb | |
| Isandiso sokugcina | 72℃ | 5 imiz | 1 |
2) Iphrothokholi Yezinyathelo Ezintathu (iphrothokholi evamile)
| Isinyathelo somjikelezo | Temp. | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10isekhondi | 30-35 |
| Anealing | 60℃ | 20 isekhondi | |
| Isandiso | 72℃ | 30 isekhondi/kb | |
| Isandiso sokugcina | 72℃ | 5 imiz | 1 |
3) I-Anealing Gradient Protocol (isifanekiso esiyinkimbinkimbi)
| Isinyathelo somjikelezo | Izinga lokushisa | Isikhathi | Imijikelezo |
| I-denaturation yokuqala | 98℃ | 3 imiz | 1 |
| I-Denaturation | 98℃ | 10 isekhondi | 15 (1℃ ukuncishiswa ngomjikelezo ngamunye) |
| Ukunciphisa i-Gradient | 70-55 ℃ | 20 isekhondi | |
| Isandiso | 72℃ | 30 isekhondi/kb | |
| I-Denaturation | 98℃ | 10 isekhondi |
20 |
| Anealing | 55℃ | 20 isekhondi | |
| Isandiso | 72℃ | 30 isekhondi/kb | |
| Isandiso sokugcina | 72℃ | 5 imiz | 1 |
Izici ngaphansi kwephrothokholi yokukhulisa ehlukile
| I-Protocol | Izinyathelo ezimbili | Izinyathelo ezintathu | Ukunciphisa i-Gradient |
| Ukucaciswa. | ngokushesha | okuphakathi | kancane |
| Ukucaciswa | phezulu | okuphakathi | phezulu |
| PCR isivuno | okuphakathi | phezulu | okuphakathi |
| Izinga lokutholwa | phezulu | okuphakathi | phezulu |
Amanothi
Sicela ugqoke i-PPE edingekayo, ijazi lelebhu kanye namagilavu, ukuze uqinisekise impilo nokuphepha kwakho!
