Author: Magold, Alexandra I.; Cacquevel, Matthias; Fraering, Patrick C.
Title: Gene Expression Profiling in Cells with Enhanced ?-Secretase Activity Document date: 2009_9_18
ID: 0p8lk12m_44
Snippet: Total RNA was reverse transcribed with our standard laboratory protocol. 1 mg of total RNA was dissolved in 4 ml of RNase-free water (Ultrapure DNase/RNase free water, Invitrogen Carlsbad, USA)) and premixed with 0.5 mg of oligo dT primer (synthesized by Eurogentec Seraing, Belgium) dissolved in 1 ml RNase-free water. The RNA/oligo dT premix was heated to 70uC for 5 minutes in a standard PCR machine (TProfessional Basic Gradient, Whatman Biometra.....
Document: Total RNA was reverse transcribed with our standard laboratory protocol. 1 mg of total RNA was dissolved in 4 ml of RNase-free water (Ultrapure DNase/RNase free water, Invitrogen Carlsbad, USA)) and premixed with 0.5 mg of oligo dT primer (synthesized by Eurogentec Seraing, Belgium) dissolved in 1 ml RNase-free water. The RNA/oligo dT premix was heated to 70uC for 5 minutes in a standard PCR machine (TProfessional Basic Gradient, Whatman Biometra Goettingen, Germany). The machine was paused to add 4 ml of 5X Buffer (ImProm-II M28A, Promega Madison USA), 4 ml of MgCl 2 (25 mM) (Promega Madison USA), 1 ml dNTP Mix (10 mM U151B, Promega Madison USA), 1 ml RNase inhibitor (RNasin Plus N261A 40 u/ul Promega Madison USA), 1 ml of ImProm-II Reverse Transcriptase (Promega Madison, USA) and 4 ml RNasefree water. The PCR machine program was continued after pausing at 25uC for completion of reaction mixes with 60 min at 42uC and 15 min at 70uC. cDNA was kept at 4uC on wet ice for short-term or at 280uC for long-term storage.
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