M-MLV Neoscript Reverse Transcriptase
I-Neoscript Reverse Transcriptase yi-reverse transcriptase efunyenwe ngokuguqula i-M-MLV gene ye-Moloney murine leukemia virus imvelaphi kunye nokubonakaliswa kwi-E.coli.I-enzyme isusa umsebenzi we-RNase H, inokunyamezela ukushisa okuphezulu, kwaye ifanelekile kwi-high-temperature reverse transcription.Ke ngoko, kuluncedo ekupheliseni iziphumo ezingathandekiyo ze-RNA yesakhiwo somgangatho ophezulu kunye nezinto ezingacaciswanga kwi-cDNA synthesis, kwaye inozinzo oluphezulu kunye nokukwazi ukuguqulela umva kokubhala.I-enzyme inozinzo oluphezulu kunye nokukwazi ukuguqulela ukuguqulwa kokubhala.
Amacandelo
1.200 U/μL Neoscript Reverse Transcriptase
2.I-5 × i-First-Strand Buffer (ukhetho)
* I-5 × i-First-Strand Buffer ayiqulathanga i-dNTP, nceda wongeze i-dNTPs xa ulungiselela inkqubo yokusabela.
Isicelo esicetyiswayo
1.Inyathelo elinye qRT-PCR.
2.Ukufunyanwa kwentsholongwane yeRNA.
Imeko yoGcino
-20°C kwindawo yokugcina ixesha elide, kufuneka ixutywe kakuhle phambi kokusetyenziswa, kunqande ukunyibilika komkhenkce rhoqo.
Inkcazelo yeyunithi
Iyunithi enye idibanisa i-1 nmol ye-dTTP kwimizuzu eyi-10 kuma-37°C isebenzisa i-poly(A)•oligo(dT)25njenge template/primer.
Ulawulo lwemeko
1.I-SDS-PAGE ukucoceka kwe-electrophoretic enkulu kune-98%.
2.Ukwandisa ubuntununtunu, i-batch-to-batch control, uzinzo.
3.Akukho msebenzi we-nuclease exogenous, akukho endonuclease exogenous okanye exonuclease ungcoliseko
Ukuseta i-Rection ye-First Chain Reaction Solution
1.Ukulungiswa komxube wokuphendula
| Amacandelo | Umthamo |
| I-Oligo(dT)12-18 Iprimer okanye I-Primer Randoma Okanye iGene Specific Primesb | 50 pmol |
| 50 pmol (20-100 pmol) | |
| 2 pmol | |
| 10 mM dNTP | 1 μL |
| Ithempleyithi ye-RNA | Iyonke i-RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-free dH2O | Ukuya kwi-10 μL |
Amanqaku:a/b: Nceda ukhethe iindidi ezahlukeneyo zeeprimers ngokweemfuno zakho zovavanyo.
2.Ukushisa kwi-65 ° C kwi-5mins kwaye upholise ngokukhawuleza kumkhenkce kwi-2mins.
3.Yongeza la malungu alandelayo kwisistim engentla kumthamo opheleleyo we-20µL kwaye uxube ngobunono:
| Amacandelo | Umthamo (μL) |
| 5 × I-First-Strand Buffer | 4 |
| I-Neoscript Reverse Transcriptase (200 U/μL) | 1 |
| I-RNase inhibitor (40 U/μL) | 1 |
| RNase-free dH2O | Ukuya kwi-20 μL |
4.Nceda wenze impendulo ngokwemiqathango elandelayo:
(1) Ukuba i-Random Primer isetyenzisiwe, ukusabela kufuneka kuqhutywe kwi-25 ℃ kwi-10mins, kwaye emva koko kwi-50 ℃ kwi-30 ~ 60mins;
(2) Ukuba i-Oligo dT okanye i-primers ethile isetyenzisiweyo, ukusabela kufuneka kuqhutywe kwi-50 ℃ ye-30 ~ 60mins.
5.Fudumeza ku-95℃ kangangemizuzu emi-5 ukuze ungasebenzi i-Neoscript Reverse Transcriptase kwaye uphelise ukusabela.
6.Iimveliso ezikhutshelwa umva zingasetyenziswa ngokuthe ngqo kwi-PCR reaction kunye ne-fluorescence quantitative PCR reaction, okanye zigcinwe ku -20℃ ixesha elide.
I-PCR Rinyathelo:
1.Ukulungiswa komxube wokuphendula
| Amacandelo | Ukugxininisa |
| 10 × PCR Buffer (dNTP simahla, Mg²+ simahla) | 1× |
| dNTPs (10mM idNTP nganye) | 200 μM |
| 25 mM MgCl2 | 1-4 mm |
| I-Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| I-Primer 1 (10 μM) | 0.2-1 μM |
| I-Primer 2 (10 μM) | 0.2-1 μM |
| Isifanekisoa | ≤10% Isisombululo sokuQala seSixokelelwano sokuQala (2 μL) |
| ddH2O | Ukuya kwi-50 μL |
Amanqaku:a: Ukuba kuninzi kakhulu isisombululo sokuqala se-chain songeziweyo, ukusabela kwe-PCR kunokuvinjelwa.
2.Inkqubo ye-PCR Reaction
| Inyathelo | Ubushushu | Ixesha | Imijikelo |
| I-denaturation yangaphambili | 95℃ | 2-5 imiz | 1 |
| I-Denaturation | 95℃ | 10-20 imizuzwana | 30-40 |
| Ukuhlaziya | 50-60℃ | 10-30 umzuzwana | |
| Ulwandiso | 72℃ | 10-60 umzuzwana |
Amanqaku
1.Ilungele ukwenziwa umva koshicilelo lobushushu kuluhlu lwe 42℃~55℃.
2.Inozinzo olungcono, ifanelekile kwiqondo lobushushu obuphezulu lokukhutshelwa kwe-reverse amplification.Ukongeza, ikulungele ukudlula ngokufanelekileyo kwimimandla entsonkothileyo ye-RNA.Kwakhona, yonailungele inyathelo elinye le-multiplex fluorescence ubhaqo RT-PCR.
3.Ukuhambelana okuhle kunye nee-enzymes ezahlukeneyo ze-PCR zokukhulisa kwaye ilungele ukusabela okuphezulu kwe-RT-PCR.
4.Ilungele uvakalelo oluphezulu inyathelo elinye le-fluorescence lobungakanani be-RT-PCR reaction, ngokufanelekileyo liphucule izinga lobhaqo loxinzelelo oluphantsi lweetemplates.
5.Ifanelekile kulwakhiwo lwethala leencwadi le-cDNA.
