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The enzyme mix used contained both β-glucanase and β-xylanase.
Fragments of the expected size were combined with an appropriate restriction enzyme mix and the appropriate enzyme buffer.
The one-step rtPCR enzyme mix used was Superscript with Platinum Taq (Invitrogen, Carlsbad, CA).
LR reactions were performed in 10 µl using 10 femtomole of each plasmid and 1 µl of LR enzyme mix (Invitrogen) or LR plus enzyme mix (multisite LR reactions, Invitrogen).
Background samples (without enzyme mix) were introduced to estimate the NADH/NADPH levels prior to G-6-P conversion.
The resultant PCR product was cloned into pDONR221 (Invitrogen) using BP Clonase II enzyme mix (Invitrogen) to generate entry clones.
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The negative control was obtained by omitting enzyme-mix from the reaction.
For M-CSFR overexpression, pEXPR clones were generated by site-specific recombination between pDEST-lenti and pENTR/D TOPO-M-CSFR by Gateway LR Clonase Enzyme mix (Invitrogen).
Each 20 µ l reaction contained 1 µg RNA, 5x RT Buffer, Primer and External Control Mix, RT Enzyme Mix (SABiosciences) and sterile ddH2O.
The PCR products were cloned into the pDONR201 entry vector using the BP Clonase™ Enzyme Mix (Invitrogen) according to the manufacturer's instructions.
These gene cassettes were introduced into pCSP mALS 43GW using MultiSite Gateway LR Clonase II Plus Enzyme Mix (Invitrogen) (Figure 2).
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