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In the case, these had been blocked, significant amounts of caffeine needed about 30 minutes to be detected [34]

The plasmid DNA was linearised with the restriction enzyme Spe1 (New England BioLabs, Inc) and transcribed into mRNA by T7 RNA polymerase with the AMBION mMESSAGE mMACHINE TM T7 transcription kit (Invitrogen TM , Thermo Fisher Scientific)

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Controlled Mixing: The mixing process must be done in a sterile, closed environment