IGEM:MIT/2006/Notebook/2006-9-16

phase 1 [Stephen]

• digest pSB1AC with EP -done
• digest WGD [resistance: a/k] (osmY.Q04400.J45119) with ES -done
• digest WGD (resistance: a/t) (J45120) with ES -done
• digest SAGD [resistance: a/t] (both 320 CA and 320 CB) with XP -done
• set up colony pcr of J45014 in yeast backbone (use special primers from samantha) -done
• start site-directed mutagenesis on all four J45398-pre mut parts -done

phase 2 [Kate]

• remove/heat shock digests -done
• PCR cleanup backbone digest -done
• make amp LCs for yeast colony PCRs -done
• ligate WGDs and SAGD with appropriate backbone! (try to find A/C) -done
• ligate ES digested FNosmy with XP cut 199 and 219 (199 and 219 are in A/K) -done
• start transformation -done

1. NOTE: Hopefully everything will work. If the transformation fails, however, here are 2 possible reasons to consider ---> the EP cut backbone post-pcr cleanup only had a concentration of 12ng/μL. Maybe something went wrong. Also, the Top10 cells that I used were ones that got thermocycled in the move to the freezer in old Endy Lab!! It is hard to find stuff up there, but I think that both of our main boxes got dumped and thermocycled. This pretty much sucks hard.

phase 3 [Andre]

• plate transformation (ready at 7pm)
• Take out colony PCR (left block) & run a gel to see if any are right (should be ~1.6kb, might be 2.6kb)

1. throw out bad LCs from 37 room (labeled 7A-H and 8A-H)

• mutagenesis dpnI digest and cleanup (right block)