链霉亲和素可以提高融合率的原因 | 吉至试剂
来自 : 蚂蚁淘
NucleotideCompositionofRNALEVELII
Materials
- RNAsample
- 1Nand0.1NHCl
- Boilingwaterbath
- Whatman#1filterpaper(forchromatography)
- Chromatographytank
- 20µlmicroPipette
- Aceticacid:butanol:water(15:60:25)solvent
- UVlightsource
- UVspectrophotometer
Procedure
- PlaceaportionofyourRNAsample(approximately40mg,hydrated)intoaheavywalledpyrextesttube.Add1.0mlof1NHClandsealthetube.
- Heatthetubeinaboilingwaterbathfor1hour.
- Coolthetube,openitandplacethecontentsintoacentrifugetube.Centrifugethecontentsat2,000RPMinaclinicalcentrifugetoremoveanyinsolubleresidue.ThesupernatantcontainsyourhydrolyzedRNA.
- PrepareWhatmanfilterpaperNo.1forstandardone-dimensionalchromatography.12
- Usingamicropipette,spot20µlofyourhydrolyzateontothepaper,beingcarefultokeepthespotsassmallaspossIBLe(repeatedsmalldropsarebetterthanonelargedrop).Allowthespotstocompletelydrybeforeproceeding.
- Placethepaperchromatogramintoyourchromatographytankandaddthesolvent(aceticacid:butanol:water).Allowthesystemtofunctionforanappropriatetime(approximately36hoursfora20cmdescendingstripofWhatman#1).Removethepaperanddryitinacirculatingairovenat40°Cforabout2hours.
- Locatethespotsofnucleotidesbytheirfluorescenceunderanultra-violetlightsource.ExposethepaperchromatogramtoaUVlightsourceandoutlinethespotsusingalightpencil.Theorderofmigrationfromthepointoforiginisguanine(lightbluefluorescence),adenine,cytilicacidandfinally,uridylicacid.
DonotlookdirectlyattheUVlightsource.UseacABInetdesignedtoshieldfromharmfulUVrADIation.
- Aftercafefullymarkingthespots,cutthemoutwithscissorsandplacethepapercutoutsintoseparatelylabeled15mlconicalcentrifugetubes.Add5.0mlof0.1NHCltoeachtubeandallowthetubestositforseveralhourstoelutethenucleotidesfromthepaper.
- Packdownthepaperwithaglassrod(centrifugeinaclinicalcentrifugeifnecessary)andremoveanaliquotoftheliquidforspectrophotometricassay.
- MeasuretheabsorbanceofeachofthefournucleotidesattheindicatedUVwavelength(havingfirstblankedtheinstrumentwith0.1NHCl).
| Base | Wavelength | MolarExtinctionCoefficient |
|---|
| Guanine | 250nm | 10.6 |
| Adenine | 260nm | 13.0 |
| Cytidylicacid | 280nm | 19.95 |
| Uridylicacid | 260nm | 9.89 |
- Usethemolarextinctioncoefficientstodeterminetheconcentrationofeachbaseinthesample.Calculatethepercentcompositionofeachbase,andthepurine/pyrimidineratio.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
