2×HiF Taq hammoho le Master Mix
Nomoro ea katse: HCR2014B
HIF Taq plus Master Mix (Ka Dye) ke tharollo e seng e loketse ho sebelisoa ea 2× premixed e nang le Plus HIF DNA Polymerase, dNTPs, le buffer e ntlafalitsoeng.Lisireletsi tse peli tsa monoclonal mocheso oa kamore tse thibelang tšebetso ea polymerase le tšebetso ea 3′→5′exonuclease li eketsoa motsoakong oa master bakeng sa Hot Start PCR e bonolo le e khethehileng haholo.Ntho e atolositsoeng e eketsoa ho motsoako o moholo ho fa enzyme matla a ho holisa likhechana tse telele, bolelele ba amplification bo ka fihla ho 13 kb, enzyme e na le ts'ebetso ea 5′→3′ DNA polymerase le 3′→5′. ts'ebetso ea exonuclease, botšepehi ba eona ke makhetlo a 83 ho feta Taq DNA polymerase, e leng makhetlo a 9 ho feta DNA polymerase e tloaelehileng.E loketse ho holisa litempele tse rarahaneng, sehlahisoa sa amplification ke pheletso e hlabang.
2 × HIF Taq plus Master Mix (With Dye) e na le melemo ea ho potlaka le bonolo, kutlo e phahameng, boits'oaro bo matla, botsitso bo botle, joalo-joalo, sistimi ea karabelo e hloka feela ho eketsa li-primers le litempele, 'me e ka holisoa ke tse peli- step protocol, ho nolofatsa mehato ea liteko le ho boloka nako.Sehlahisoa sena se na le lidae tsa electrophoresis indicator, 'me lihlahisoa tsa PCR li ka sebelisoa ka ho toba bakeng sa electrophoresis.Ho phaella moo, sehlahisoa se boetse se na le ntho e itseng e sireletsang, e le hore motsoako o ka sehloohong o ka boloka ts'ebetso e tsitsitseng ka mor'a ho qhibiliha khafetsa.
Maemo a polokelo
Lihlahisoa li lokela ho bolokoa ho -25 ~ -15 ℃ bakeng sa selemo se le seng.
Litlhaloso
| Tlhaloso ea lihlahisoa | Master Mix |
| Ho tsepamisa maikutlo | 2× |
| Hot Qala | E hahiloeng ka Hot Start |
| Overhang | Bohlasoa |
| Lebelo la ho arabela | Ka potlako |
| Boholo (Sehlahisoa sa ho Qetela) | Ho fihlela ho 13kb |
| Maemo a lipalangoang | Leqhoa le omileng |
| Mofuta oa sehlahisoa | Li-premixes tsa PCR tse tšepahalang haholo |
Litaelo
1.Sistimi ea Karabelo ea PCR
| Likaroloana | Bolumo (μL) |
| Setšoantšo sa DNA | E loketse |
| Sehlahisoa sa pele (10 μmol/L) | 2.5 |
| Primer e khutlisang (10 μmol/L) | 2.5 |
| 2×HIF Taq hammoho le Master Mix | 25 |
| ddH2O | ho ea ho 50 |
2.Tšebeliso e khothalletsoang ea litempele tse fapaneng
| Mofuta oa template | Matlafatsa likhechana ho tloha ho 1kb ho isa ho 10 kb |
| Genomic DNA | 50ng-200 ng |
| Plasmid kapa Viral DNA | 10pg-20ng |
| cDNA | 1-2.5 µL (Se ke oa feta 10% ea molumo oa ho qetela oa karabelo ea PCR) |
3.Amplification Protocol
1) Protocol ea Mehato e 'meli (sebopeho se rarahaneng)
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Denaturation ea pele | 98℃ | 3 mets | 1 |
| Denaturation | 98℃ | 10sec | 30-35 |
| Katoloso | 68℃ | 30 sec/kb | |
| Katoloso ea ho qetela | 72℃ | 5 mets | 1 |
2) Protocol ea Mehato e meraro (protocol e tloaelehileng)
| Mohato oa potoloho | Temp. | Nako | Lipotoloho |
| Denaturation ea pele | 98℃ | 3 mets | 1 |
| Denaturation | 98℃ | 10sec | 30-35 |
| Annealing | 60 ℃ | 20 metsotsoana | |
| Katoloso | 72℃ | 30 sec/kb | |
| Katoloso ea ho qetela | 72℃ | 5 mets | 1 |
3) Annealing Gradient Protocol (sebopeho se rarahaneng)
| Mohato oa potoloho | Mocheso | Nako | Lipotoloho |
| Denaturation ea pele | 98℃ | 3 mets | 1 |
| Denaturation | 98℃ | 10 sec | 15 (Phokotso ea 1 ℃ potoloho ka 'ngoe) |
| Ho fokotsa likhahla | 70-55 ℃ | 20 metsotsoana | |
| Katoloso | 72℃ | 30 sec/kb | |
| Denaturation | 98℃ | 10 sec |
20 |
| Annealing | 55℃ | 20 metsotsoana | |
| Katoloso | 72℃ | 30 sec/kb | |
| Katoloso ea ho qetela | 72℃ | 5 mets | 1 |
Lintlha tse tlas'a protocol e fapaneng ea amplification
| Protocol | Mehato e 'meli | Mehato e meraro | Ho fokotsa likhahla |
| Spec. | ka potlako | mahareng | butle |
| Tse khethehileng | hodimo | mahareng | hodimo |
| Setšoantšo sa PCR | mahareng | hodimo | mahareng |
| Sekhahla sa ho lemoha | hodimo | mahareng | hodimo |
Lintlha
Ka kopo, roala PPE e hlokahalang, jase e joalo ea lab le liatlana, ho netefatsa bophelo bo botle le polokeho ea hau!
