I-RT-LAMP Fluorenscent Master Mix
Lo mkhiqizo uqukethe isigcinalwazi sokusabela, i-RT-Enzymes Mix (i-Bst DNA polymerase kanye ne-reverse transcriptase engashisi ukushisa), ama-Lyophilized Protectants kanyei-fluorescentizingxenye zodayi.Ibhafa yokusabela iqukethe i-Mg2+, i-dNTP nezinye izingxenye ezidingekayo zokukhulisa.Ukuze usebenzise, vele uhlanganise i-Buffer, i-enzyme yokusabela, udayi we-fluorescent, i-primer bese wengeza isifanekiso, ukwengeza isivikeli se-lyophilized singasebenzisa ngokuqondile.Yayixhunywe ku-lyophilizer kanye ne-lyophilized, futhi kuphela ama-primers kanye nezifanekiso zengezwe lapho zisetshenziswa.Lesi sici sinokwethenjelwa okuphezulu kwe-amplification kanye nokuzwela.
Isakhi
Isakhi | I-HCB5205A-01 | HCB5205A-02 | HCB5205A-03 |
Ibhafa yokukhulisa i-Loop-mediated | 1.2 mL | 4 mlx3 | 12 mLx10 |
I-RT-Enzymes Mix | 245 μL | 2.45 mL | 2.45 mL×10 |
Isivikelo se-Lyophilized | 0.96 mL×2 | 9.60 mL×2 | 9.60 mL×20 |
25× Udayi | 192 μL | 0.96 mL×2 | 0.96 mL×20 |
Izinhlelo zokusebenza
Nge-DNA noma i-RNA isothermal amplification.
Izimo Zokugcina
Kuthuthwa ngeqhwa elomile, eligcinwe ku -25~ -15 ℃.Gwema ukuncibilika kweqhwa njalo, umkhiqizo usebenza izinyanga eziyi-12.
Iphrothokholi
1.Ncibilikisa isigcinalwazi esizosetshenziswa ekamelweni lokushisa.I-Vortex kafushane noma guqula amashubhu izikhathi ezimbalwa ukuze ixube kahle, bese i-centrifuge ukuqoqa uketshezi phansi kweshubhu.
2.Ukulungiswa kwesistimu yokusabela.Lesi sici singalungiswa ngezinhlelo ezimbili zokusabela, ingxube ye-liquid reaction kanye ne-lyophilized system mix.
1) Lungiselela ukuxubana kwe-liquid reaction
Isakhi | Ivolumu |
Ibhafa yokukhulisa i-Loop-mediated | 12.5 μL |
25× Udayi | 2 ml |
I-RT-Enzymes Mix | 2.55 μL |
10 × I-Primer Mixa | 5 μl |
Izifanekiso ze-DNA/RNA b | × μL |
Amanzi Angenayo I-nuclease | Kufika ku-50 μL |
2) I-Lyophilization system mix
① Lungiselela imiksi ye-lyophilized
Isakhi | Ivolumu |
Ibhafa yokukhulisa i-Loop-mediated | 12.5 μL |
Isivikelo se-Lyophilized | 20 μL |
25× Udayi | 2 ml |
I-RT-Enzymes Mix | 2.55 μL |
Amanzi Angenayo I-nuclease | Kufika ku-50 μL |
② I-Lyophilization: I-Mix elungisiwe i-lyophilized ohlelweni lwe-50μL
③ Lungiselela imiksi yokusabela
Isakhi | Ivolumu |
Ingxube ye-Lyophilized | 1 isiqephu |
10 × I-Primer Mixa | 5 μl |
Izifanekiso ze-DNA/RNA b | × μL |
Amanzi Angenayo I-nuclease | Kufika ku-50 μL |
Amanothi:
1) a.10×I-Primer Mix : 16 μM FIP/BIP, 2 μM F3/B3, 4 μM Iluphu F/B;
2) b.I-DEPC (i-soluble yamanzi) inconywa nge-nucleic acid templ.
3)Ukusabela Kokukhulisa: Setha uhlelo olulandelayo ensimbini ye-PCR ekhanyayo (isb. i-ABI7500, njll.):
Izinga lokushisa | Isikhathi | Imibuthano |
65 ℃ | 60 isekhondi *qoqa i-FAM fluorescence | 30 |
Amanothi
1.Usawoti ungase uvele ngaphansi kweshubhu lebhafa, i-vortex kafushane noma uguqule amashubhu izikhathi ezimbalwa ukuze uxubane kahle ekamelweni lokushisa.
2.Izinga lokushisa lokusabela lingenziwa kahle phakathi kuka-62 ℃ no-68 ℃ ngokwesimo sezinto zokuqala.
3.Ukuhlolwa kufanele kufane, okuhlanganisa ukulungiswa kwesistimu yokusabela, i-lyophilization, nokucubungula isampula kanye nenqubo yokwengeza isampula;
4.Ukuze ugweme ukungcola, kunconywa ukuthi ulungise isistimu yokusabela ebhentshini elihlanzeke kakhulu, kwelinye Engeza izifanekiso ku-fume hood yegumbi ukuze ugweme ukugxambukela okungamanga.