1. PCR Product Orientation Test T7 EcoRI Cloning Site EcoRI PmeI PstI SpeI NotI pCR4-TOPO T7

2. PCR ProductT7 EcoRI PmeI PstI SpeI NotI T7 PCR ProductT7 EcoRI PmeI PstI SpeI NotI T7 Effective Orientation Ineffective Orientation

3. PCR ProductT7 EcoRI PmeI PstI SpeI NotI T7 PCR ProductT7 EcoRI PmeI PstI SpeI NotI T7 Effective Orientation Ineffective Orientation SpeI Digest SpeI Vector (~4000bp) + Small PCR Frag Digest SpeI Vector (~4000bp) + Larger PCR Frag

4. HT115 DE3 E. coli Strain –Null mutation of rnc null (RNaseIII) RNase III degrades double stranded RNA –DE3 – lambda lysogen containing IPTG inducible T7 RNA polymerase Lysogen contains tet resistance gene Timmons L, Court DL, Fire A (2003)

5. Transformation Wednesday –Add Tetracyline to LB broth –Inoculate with culture of HT115 DE3 –Grow O/N 37° Thursday AM –Prepare second flask of LB+tet –Inoculate with 500ml of overnight culture HT115DE3. –Grow ~4 hours Thursday PM –Treat Cells with CaCl to make competent –Mix cells with RNAi vector –Spread on LB + tet + amp plates –Grow O/N 37°