IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/04

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Wet Work

  • Check vectors

- for each vector, plasmid miniprep from frozen pellets (step 7 only once, with 60μL of Elution Buffer for ECE112, ECE147, ECE149, ECE 150 and only 30μL for the others)

- Single digest (15μL of SDW, 2μL of Buffer, 2μL of DNA, 1μL of enzyme, then incubate 10min at 37°C and 30 min for the one using HindIII with EcoRI buffer)

- Double digest (mix 7.5μL of each single digest, and then incubate 10min (or 30min for EcoRI + HindIII and EcoRI buffer) at 37°C, then heat shock 5min at 80°C

Vectors Enzyme 1 Enzyme 2 Buffer Entire size Size of part1 Size of part2
ECE112 XbaI EcoRI Fast Digest 10156 6900 3295
ECE147 EcoRI HindIII EcoRI 5482 4913 600
ECE149 SpeI PstI Fast Digest 6192 4919 1300
ECE150 SpeI HimdIII Buffer 2 6166 5300 778
ECE151 SpeI EcoRI Fast Digest 6166 4825 1300
ECE153 SalI Buffer 3
ECE162 SalI Buffer 3 7600 6000 1600
ECE165 EcoRI HindIII EcoRI 5952 5200 793
ECE166 EcoRI HindIII EcoRI 7262 5100 2103
ECE171 EcoRI PstI Fast Digest 5444 3600 1826
ECE172 HindIII Buffer 2 6462 3900 2540
ECE176 EcoRI XbaI Fast Digest 866 4250 5128
  • Results
  • Lane1 : HyperladderI
  • Lane2 : ECE112
  • Lane3 : ECE147
  • Lane4 : ECE149
  • Lane5 : ECE150
  • Lane6 : ECE151
  • Lane7 : ECE162
  • Lane8 : ECE165
  • Lane9 : ECE166
  • Lane10 : ECE171
  • Lane11 : ECE172
  • Lane12 : HyperladderI


- ECE112, ECE165, ECE166, ECE171 : right bands, ok

- ECE172 : no band, not enough DNA

- ECE147, ECE150, ECE162 : only one band, maybe not enough DNA

- ECE149 : wrong size!

- ECE151 : not sure, check again


  • Plates

- Streak new plates with different strains of Bacillus : 1A1, IA751 and IA771


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