IGEM:MIT/2006/Notebook/2006-8-2

to do

1. LC B0030
2. LC R0040
3. LC B0015
4. LC ATF1.B0015 variants
5. LC R0040.E0840

1. analyze 42 sequence results using prepared contigs in VectorNTI -done. unfortunately our analysis shows that nothing seems to have inserted. Perhaps digests are not being run long enough? also, pchBA mutagenesis was unsuccessful.
2. start isoamyl alcohol generating device work
   • PCR BAT2 and THI3 again to get higher DNA concentrations for digesting/ligating on 8/3 -done
3. (3 pm) meeting with advisors -done
4. run gel of PCR products/PCR cleanup and nanodrop BAT2 and THI3
5. colony PCRs -done
6. make liquid cultures from each potentially useful transformant plate and glycerol stocks for assembly on 8/3

meeting topics

1. troubleshoot pchBA primers
2. troubleshoot pchBA mutagenesis
3. troubleshoot digests/failed assemblies
4. timeline of assembly work (best case scenario)

liquid cultures from potentially useful plates

1. 7/31 plates: 4, 5, 6, 7, 8
   • 4/5/6/7 = maybe ATF1mut-Term (colony PCR to check first)
   • 8 = maybe ROO40-E0840
2. 8/1 plates: 7, 8, 9
   • 7/8/9 = maybe ATF1mut-Term (colony PCR to check first)

primers

1. design/order THI3 mutagenesis primers
2. order new (add 2 more annealing bases) pchA reverse primer

team meeting: assembly plan

1. current status of parts:
   • WGGD: BSMT+Terminator
   • SAGD: still trying to PCR pchBA
   • BSGD: (?) ATF1mut+Term (?)
   • IAGD: PCRed both coding regions
   • osmY: pcr product