Details
Source
Thermusaquaticus
Description
- Thermostableenzymeofapproximately94kDafrom Thermusaquaticus
- Ultrapure,Recombinantprotein
Applications
- RecommendedforuseinPCRandprimerextensionreactionsatelevatedtemperaturestoobtainawiderangeofDNAproducts upto10Kb
- TaqDNAPolymeraseenzymereplicatesDNAat74°Candexhibitsahalf-lifeof40minutesat95°C(1,2)
- TaqDNAPolymerasecatalyzesthepolymerizationofnucleotidesintoduplexDNAinthe5"→3"directioninthepresenceofmagnesiumions
- Maintainsthe5"→3"exonucleaseactivity
- Lacksthe3"→5"exonucleaseactivity
ReagentsSupplied
10XTaqBufferA
10XTaqBufferB
10XTaqBufferC
UnitDefinition
Oneunitisdefinedastheamountofenzymerequiredtocatalyzetheincorporationof10nmolesofdNTPintoacid-insolublematerialin30minutesat70°C
10XReactionBuffer
10XTaqBufferA(optimizationbufferwithoutMgCl2):BufferallowstooptimizeMgCl2 concentration
10XTaqBufferB(generalapplication,upto10kb):Buffercontains15mM MgCl2 andis optimizedforusewith0.2 mMofeachdNTP
10XTaqBufferC(colored): 10XTaqBufferBEnrichedwithtwogeltrackingdyesandagelloADIngreagent;EnablesdirectloadingofPCRproductsontoanagarosegel
StorageBuffer
20mMTris-HCl(pH8.0at22°C)
100mMKCl
0.1mMEDTA
1mMdithiothreitol
50%glycerol
StABIlizers
AssayConditions
25mMTris-HCl(pH9.5at25°C)
50mMKCl
10mMMgCl2
1mMdithiothreitol
200μMeachofdCTP,dGTP,dTTP,anddATP(amixofunlabeledand[α-32P]dATP)
10μgactivatedcalfthymusDNA
1mg/mlbovineserumalbumin
15 µgactivatedcalfthymusDNA
Totalreactionvolumeis50 µl
QualityControl
Allpreparationsareassayedforcontaminatingendonuclease,3"-exonuclease,andnonspecificsingle-anddouble-strandedDNaseactivities
Typicalpreparationsaregreaterthan95%pure,asjudgedbySDSpolyacrylamidegelelectrophoresis
StorageConditions
Storeat-20°C
Shippedondryice
Downloads
MSDSPDF
References
1.Chien,A.,Edgar,D.B.andTrela,J.M.(1976)J.Bacteriol.127,1550
2.Kaledin,A.S.,Sliusarenko,A.G.andGorodetskii,S.I.(1980)Biokhimiya45,644
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希望同路人多多交流啊!
这些染料都非常成熟,光毒和淬灭都很低,当然要考虑到你采集图像时的显微镜参数。
比如calcein,常用的示踪,绿光(虽然这些颜色只是根据光谱加上去的伪彩),但要考虑你的实验过程中结合细胞结构,是否会伴随calcein的泄漏,就是荧光降低。
dri,还可以看膜啊
cfda也不错
bcef虽是ph指示,但你试验时不仅可观察细胞,还可看细胞ph变化,也行。
当然你或许还要结合其他方法,如细胞免疫化学等手段去双染或多染,都需要综合考虑染料之间特性。单独染一个染料,有点浪费,不如多染,数据和图像也好看些。现在流行细胞成像。

