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Tuesday 10/19/10 we digested our promoter BBa_i12007, double terminator BBa_B0015 to prepare for future ligation. We also re digested our gene BBa_E1010 due to improper cutting with restriction enzymes, the first time due to a change in our final ligation steps. Digested the promoter with enzymes spe1-pst1, the double terminator with EcoRl-Xball and our gene with spe1-EcoR1. Also we ran our gene and digested on a gel, cut out for purification later.
Thursday 10/21/10 we used the PCR machine on our two combined oligo’s TUNA & TINTIN to annneal them. Cycle ran starting at 70C decreasing .5C every 30sec, down to 40C. 80 total cycles. Was done to prepare RBS for ligation to promoter. Also did a gel purification using GeneJet gel extraction kit on our Gene. Purified our double terminator using GeneJet PCR purification kit. We did not purify our promoter piece due to wrong enzyme cut location. Next time we will re digest just cutting with spe1 making it possible for our RBS piece to bind.