FAQ: If I accidentally move all 30 ul of the 5’ ligation product forward to the reverse transcription mixture will the reaction still work?

Moving all of the 5’ ligation reaction product into the reverse transcription reaction will greatly reduce the activity of the M-MuLV (RNase H-) Reverse Transcriptase due to the elevated levels of Mg. Amplification output will be only ~15% of what would have been attained with 14 ul of 5’ ligation product.