To correct enhancer activity for the amount of read-through that is potentially generated from the enhancer TSS, we additionally generated constructs lacking the basal EF1α promoter for all B cell-specific constructs. Relative luciferase activities generated by read-through activity were subtracted from the activity of enhancer/EF1 constructs to reveal ‘true’ enhancer activities of individual regions. To further determine the position and activity of reporter TSS, 5’ RACE-PCR for the luciferase gene was performed as follows: RNA of transfected DAUDI cells was reverse transcribed using the SMARTer™ RACE cDNA Amplification Kit (Clontech, France) according to the manufacturers’ instructions. Rapid Amplification of luciferase 5’ cDNA ends (5’ RACE) was performed with the Advantage 2 Polymerase System (Clontech) and a LUC specific primer (5’-CAT GGC TTC TGC CAG CCT CAC AGA CAT C-3’) using the recommended touchdown-PCR program. 15μl of the PCR products were analyzed by agarose gel electrophoresis (2.5%). In addition, fragments were cloned using the StrataClone PCR cloning Kit (Agilent) according to the manufacturers’ instructions and sequenced (Life Technologies, Germany).
