IGEM:MIT/2006/Notebook/2006-8-2: Difference between revisions

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==to do==
==to do==
#(am) analyze 42 sequence results using prepared contigs in VectorNTI
#LC B0030
#LC R0040
#LC B0015
#LC ATF1.B0015 variants
#LC R0040.E0840
 
<br><br>
#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.
#start isoamyl alcohol generating device work
#start isoamyl alcohol generating device work
#'''prepare topics for meeting at 3 pm'''
#*PCR BAT2 and THI3 again to get higher DNA concentrations for digesting/ligating on 8/3 -'''done'''
#*ask for help with pchA reverse primer (???)
#(3 pm) meeting with advisors -'''done'''
#*assess project progress/timeline
#run gel of PCR products/PCR cleanup and nanodrop BAT2 and THI3
#order new pchA reverse primer
#colony PCRs -'''done'''
#test osmY-E0840 with plate reader
#make liquid cultures from each potentially useful transformant plate and glycerol stocks for assembly on 8/3
#(pm) make liquid cultures A,B,C from each transformant plate (pending colonies and promising sequence analysis)
 
#*note possible 2/4/9 mix-up and that 9 was an A/K plate!!! so presence/absence of colonies may help us figure things out better.
==meeting topics==
#troubleshoot pchBA primers
#troubleshoot pchBA mutagenesis
#troubleshoot digests/failed assemblies
#timeline of assembly work (best case scenario)
 
==liquid cultures from potentially useful plates==
#7/31 plates: 4, 5, 6, 7, 8
#*4/5/6/7 = maybe ATF1mut-Term ('''colony PCR to check first''')
#*8 = maybe ROO40-E0840
#8/1 plates: 7, 8, 9
#*7/8/9 = maybe ATF1mut-Term ('''colony PCR to check first''')
 
==liquid cultures from glycerols for assembly 8/3==
 
==primers==
#design/order THI3 mutagenesis primers
#order new (add 2 more annealing bases) pchA reverse primer
 
==team meeting: assembly plan==
#current status of parts:
#*WGGD: BSMT+Terminator
#*SAGD: still trying to PCR pchBA
#*BSGD: (?) ATF1mut+Term (?)
#*IAGD: PCRed both coding regions
#*osmY: pcr product

Latest revision as of 15:03, 2 August 2006

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)

liquid cultures from glycerols for assembly 8/3

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
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