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


 * 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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