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Shrimp alkaline phosphatase or SAP, is an enzyme which catalyzes the dephosphorylation of 5’ phosphates from nucleic acid dephosphorylating RNA or DNA ends. Kepec alkalna fosfataza ili SAP je enzim koji catalyzes je dephosphorylation od 5 'fosfati iz nucleic acid dephosphorylating RNA ili DNA završava.
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! Imajte na umu da PCR proizvoda i početnice nemaju phosphatases na svoje slobodno kraja, i stoga trebate biti phosphorylated prije klon, ako ćete koristiti de-phosphorylated vektor!
SAP is temperature unstable are therefore you can easily deactivate this enzyme (compared to CIP!). SAP je temperatura nestabilni su, dakle, možete jednostavno prestati ovaj enzim (u odnosu na CIP!). SAP is deactivated with 65°C water bath incubation for 15 minutes. SAP je deaktiviran s 65 ° C vodenoj kupki inkubacije za 15 minuta.
You get virtually no reaction. Dobivate gotovo nema reakcije. 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 zahtijeva najmanje 10mm MgCl2 (ali optimalnu aktivnost kepec enzim alkalna fosfataza je na 20-25mm MgCl2) i to dobro radi na pH 8,0.
To dephosphorylate 1 pmol of DNA ends (2.5 ug of 3 Kb plasmid), for 1hr at 37°C you need: Da dephosphorylate 1 pmol DNA završava (2,5 ug od 3 Kb plasmid), za 1 h na 37 ° C što Vam treba:
0.1 units of SAP for 5' overhangs, 0.2 units for blunt and 0.5 units for 3' over hangs. 0,1 jedinice SAP za 5 'overhangs, 0,2 jedinica za tupim i 0,5 jedinica za 3' nad objesi.
Enzyme comes 1 unit per ul, Thus to be safe you can use approx.0.5 units for 2 ug. Enzim dolazi 1 jedinica po ul., čime bi se sigurno možete koristiti approx.0.5 jedinice za 2 ug.
TIPS: SAP Enzyme can be added directly to restriction digest! SAVJETI: SAP Enzyme može biti dodano izravno na ograničenje sažeto! 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. Ovako cijeli postupku, uključujući ograničenje enzim probavu, dephosphorylation, enzim deaktiviranje, ligation ili 5 'end-označavanje može biti izvedena u jednu cijev uz samo dodavanjem odgovarajućeg Reagensi. This will allow you to save time and be efficient! Ovo će vam omogućiti da uštedite vrijeme i biti učinkovita!
Discuss and post questions about it in the Shrimp Alkaline Phosphatase and DNA Cloning Forum ! Diskusiji i postavljati pitanja o tome u ništavilo Alkaline fosfataza i DNA Cloning Forum!
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