home > dna > sap-shrimp-alkaline-phosphatase-protocol > index.php home> DNA> SAP-reje-alkalisk-fosfatase-protokol> index.php
 |
|---|
You have to register before you can post on our forums or use our advanced features. Du er nødt til at registrere før du kan skrive indlæg på vores fora eller brug vores avancerede funktioner. Register Now! Tilmeld dig nu! Its Free and Fast! Dens frie og hurtigt!
Already registered? Allerede registreret? Login now below. Login nu nedenfor.
Already registered and Forgot your password? Allerede registreret og Har du glemt dit password? Click below to recover it. Klik nedenfor for at genoprette den.
Recover Lost Password Recover Lost Password
Join now - it's fast and free! Tilmeld dig nu - det er hurtigt og gratis!
Molecular Station is THE largest network of researchers, scientists and science lovers anywhere! Molekylær Station er det største netværk af forskere, videnskabsmænd og videnskab kærester overalt!
The great men of science are supreme artists. Den store mænd af videnskab er øverste kunstnere. ~Martin H. Fischer ~ Martin H. Fischer
Shrimp alkaline phosphatase or SAP, is an enzyme which catalyzes the dephosphorylation of 5’ phosphates from nucleic acid dephosphorylating RNA or DNA ends. Rejer alkalisk fosfatase eller SAP, er et enzym, der catalyzes de dephosphorylation af 5 'fosfater fra nukleinsyre dephosphorylating RNA eller DNA ender.
Note that PCR products and also primers do not have phosphatases on their free ends, and therefore need to be phosphorylated prior to cloning if you are going to use a de-phosphorylated vector! Bemærk, at PCR-produkter og også primere ikke har phosphatases på deres frie ender, og derfor skal fosforyleret forud for kloning, hvis du vil bruge en de-fosforyleret vektor!
SAP is temperature unstable are therefore you can easily deactivate this enzyme (compared to CIP!). SAP er temperaturen ustabil er derfor du nemt kan deaktivere dette enzym (i forhold til CIP!). SAP is deactivated with 65°C water bath incubation for 15 minutes. SAP er deaktiveret med 65 ° C vandbad inkubation i 15 minutter.
You get virtually no reaction. Du får næsten ingen reaktion. SAP requires at least 10mM MgCl2 (but optimal enzyme activity of shrimp alkaline phosphatase is at 20-25mM MgCl2) and it works well at pH 8.0. SAP kræver mindst 10mm MgCl2 (men optimal enzym aktivitet af rejer alkalisk fosfatase er på 20-25mm MgCl2), og det fungerer godt ved pH 8,0.
To dephosphorylate 1 pmol of DNA ends (2.5 ug of 3 Kb plasmid), for 1hr at 37°C you need: At dephosphorylate 1 pmol af DNA ender (2,5 ug af 3 Kb plasmid), for 1 t ved 37 ° C, skal du bruge:
0.1 units of SAP for 5' overhangs, 0.2 units for blunt and 0.5 units for 3' over hangs. 0,1 enheder af SAP for 5 'overhangs, 0,2 enheder for sløv og 0,5 enheder til 3' over hangs.
Enzyme comes 1 unit per ul, Thus to be safe you can use approx.0.5 units for 2 ug. Enzyme kommer 1 enhed pr ul, således at være sikker kan du bruge approx.0.5 enheder til 2 ug.
TIPS: SAP Enzyme can be added directly to restriction digest! TIPS: SAP Enzyme kan tilføjes direkte til begrænsning fordøje! Thus the whole proceeding including restriction enzyme digestion, dephosphorylation, enzyme inactivation, ligation or 5’ end-labeling can be performed in one single tube by just adding the appropriate reagents. Således er hele proceduren, herunder begrænsning enzym fordøjelse, dephosphorylation, enzym inaktiveres, ligation eller 5 'end-labeling kan udføres i et enkelt rør ved blot at tilføje et passende reagenser. This will allow you to save time and be efficient! Dette vil give dig mulighed for at spare tid og være effektivt!
Discuss and post questions about it in the Shrimp Alkaline Phosphatase and DNA Cloning Forum ! Diskutere og stille spørgsmål om det i rejer alkalisk fosfatase og DNA Kloning Forum!
DNA Cloning Guide DNA Kloning Guide
DNA Gel Electrophoresis DNA gelelektroforese
Restriction Enzyme Digestion - all you need to know ! Restriction Enzyme Fordøjelsesprocedure - alt du behøver at vide!
Genomic DNA Isolation Protocol Genomisk DNA Isolering protokol
Baceteria DNA Isolation Baceteria DNA Isolering
DNA Transformation - Contains History of DNA DNA Transformation - Indeholder History of DNA
Find other protocols at our DNA Protocols external resource directory or see our DNA protocols . Find andre protokoller på vores DNA protokoller ekstern ressource bibliotek eller se vores DNA-protokoller.
Related:
DNA Microarray Protocols DNA microarray protokoller
Visit DNA Bioinformatics . Besøg DNA bioinformatik.
Disclaimer / Terms of Service | Privacy Policy | ©2005-2007 Molecular Station.com, All rights reserved. Disclaimer / Terms of Service | Privacy Policy | © 2005-2007 Molekylær Station.com, Alle rettigheder forbeholdt.
Send this page to a friend Send denne side til en ven
![[أربيك]](http://www.molecularstation.com/translate/images/flag_ar.gif){kind=small}
