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== Adaptation to totoRNAseq == *Test: **Ambion PAP vs Enzymatics PAP in RT/RNaseIII buffer ***Commercial mix Ambion (see above) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP ***totoRNAseq mix Ambion (below) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP ***totoRNAseq mix Enzymatics (below) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP :'''totoRNAseq Reaction Buffer''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |1uM TSO.r04||||||||||1ul |- |dH20||||||||||6.4ul |- |5x RT buffer||||||||||1.25ul |- |10x RnaseIII buffer||||||||||0.5ul |- |1mM ATP or 10mM ATP||||||||||0.625ul |- |0.5U PAP||||||||||0.25ul |- |} [[File:10232013_gel4.jpg]] Result: Unlike in commercial buffer, Ambion PAP and Enzymatics PAP were not inhibited by 3'dATP in totoRNAseq buffer. totoRNAseq buffer has too high of Mg levels - uncontrolled PAP activity Ambion 1x buffer [50mM Tris; 0.25M NaCl2; 2.5mM MnCl2] RNaseIII 1x buffer [10mM Tris; 10mM MgCl2; 1mM DTT; 60mM NaCl] MMLV RT Buffer [30mM Mgcl2...] totoRNAseq buffer [8.75mM total MgCl2) *Test: **Ambion PAP without RT buffer ***Commercial mix Ambion (see above) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP ***totoRNAseq mix -RT (below) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP ***totoRNAseq mix -RT +NaCl (below) ****10mM ATP ****1mM ATP ****1mM ATP/0.1mM 3' d-ATP ****1mM ATP/0.5mM 3' d-ATP :'''totoRNAseq Reaction Buffer''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |1uM TSO.r04||||||||||1ul |- |dH20||||||||||7.625ul (6.525 + 1.1ul 2M NaCl) |- |10x RnaseIII buffer||||||||||0.5ul |- |ATP||||||||||0.625ul |- |0.5U PAP||||||||||0.25ul |- |} [[File:10232013_gel5.jpg]] Note: Best PAP activity is in the Commercial buffer conditions - therefore may need to determine whether RNAseIII can still digest under PAP buffer conditions...
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