I-Bst 2.0 DNA Polymerase (iGlycerol free)
I-Bst DNA polymerase V2 isuselwa ku-Bacillus stearothermophilus DNA Polymerase I, enomsebenzi we-5′→3′ DNA polymerase kanye nomsebenzi oqinile wokushintsha iketango, kodwa akukho 5′→3′ umsebenzi we-exonuclease.I-Bst DNA Polymerase V2 ifaneleka ngokufanelekile ukususwa kwe-strand, i-LAMP yokukhulisa i-isothermal (i-Loop mediated isothermal amplification) nokulandelana ngokushesha.
Izingxenye
| Isakhi | I-HC5005A-01 | I-HC5005A-02 | I-HC5005A-03 |
| I-BstDNApolymerase V2(Glycerol-free)(8U/μL) | 0.2 mL | 1 mL | 10 ml |
| 10×HC Bst V2 Buffer | 1.5 mL | 2×1.5 mL | 3 × 10 mL |
| MgSO4(100mM) | 1.5 mL | 2×1.5 mL | 2 × 10 mL |
Izinhlelo zokusebenza
1.LAMP isothermal amplification
2.DNA strand single displacement reaction
3.High GC gene ukulandelana
4.Ukulandelana kwe-DNA kwezinga le-nanogram.
Isimo Sesitoreji
Ukuthutha ngaphansi kuka-0°C futhi kugcinwe ku -25°C~-15°C.
Incazelo Yeyunithi
Iyunithi eyodwa ichazwa njengenani le-enzayimu elihlanganisa u-25 nmol we-dNTP kwinto engancibiliki ene-asidi emizuzwini engama-30 ku-65°C.
Ikhwalithi yokulawula
1.I-Protein Purity Assay (SDS-PAGE):Ukuhlanzeka kwe-Bst DNA polymerase V2 kungu-≥99% kunqunywa ukuhlaziya kwe-SDS-PAGE kusetshenziswa ukutholwa kwe-Coomassie Blue.
2.Umsebenzi we-Exonuclease:Ukufakwa kokusabela okungu-50 μL okuqukethe ubuncane obungu-8 U we-Bst DNA polymerase V2 ene-1 μg λ -Hind Ⅲ digest DNA amahora angu-16 ngo-37 ℃ akuphumeleli ukuwohloka okubonakalayo njengoba kunqunyiwe.
3.Umsebenzi we-Nickase:Ukufakwa kokusabela okungu-50 μL okuqukethe ubuncane be-8 U ye-Bst DNA polymerase V2 ene-1 μg pBR322 DNA amahora angu-16 ku-37°C akuphumeleli ukuwohloka okubonakalayo njengoba kunqunyiwe.
4.Umsebenzi we-RNase:Ukufukamela kokusabela okungu-50 μL okuqukethe ubuncane obungu-8 U we-Bst DNA polymerase V2 ene-1.6 μg MS2 RNA amahora angu-16 ku-37°C kubangela ukungabikho kokuwohloka okubonakalayo njengoba kunqunyiwe.
5.E. coli DNA:I-120 U ye-Bst DNA polymerase V2 ihlolelwa ukuba khona kwe-E. coli genomic DNA kusetshenziswa i-TaqMan qPCR eneziqalo eziqondile ze-E. coli 16S rRNA locus.Ukungcola kwe-E. coli genomic DNA kungu-≤1 Ikhophi.
I-LAMP Reaction
| Izingxenye | 25μL |
| 10×HC Bst V2 Buffer | 2.5 μL |
| MgSO4 (100mM) | 1.5 μL |
| Ama-dNTP (10mM lilinye) | 3.5 μL |
| I-SYTO™ 16 eluhlaza (25×)a | 1.0 μL |
| I-Primer mixb | 6 ml |
| I-Bst DNA Polymerase V2 (Glycerol-free) (8 U/uL) | 1 μl |
| Isifanekiso | × μL |
| ddH₂O | Kufika ku-25 μL |
Amanothi:
1) a.I-SYTOTM 16 Green (25×): Ngokwezidingo zokuhlola, amanye amadayi angasetshenziswa njengama-substitutes;
2) b.Ingxube yokuqala: itholwe ngokuxuba 20 µ M FIP, 20 µ M BIP, 2.5 µ M F3, 2.5 µ M B3, 5 µ M LF, 5 µ M LB namanye amavolumu.
Ukusabela kanye Nesimo
1 × HC Bst V2 Buffer, izinga lokushisa ekufukameleni liphakathi kuka-60°C no-65°C.
Ukushisa Ukungasebenzi
80 ° C, imizuzu engu-20
