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