After my earlier reaction it is time to continue on. I have purified the ligation reactions from before to remove enzyme and unligated adapter molecules (QiaQuick cleanup kits work really well for purifying short DNA molecules). I ran some samples in the nanodrop to calculate the resulting concentrations (shown in the reaction below, and calculated from the DNA concentration calculator). Next I’m running the reaction below:

• 5pBR – pBR cut with SapI and ligated to the 5′-bio adapter
• IpBR – pBR cut with SapI and ligated to the internal-bio adapter
• pRL anchor – 1.1kb PCR fragment obtained from pRL574 and cut with BstXI