IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/04

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==Wet Work==
 
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* '''Check vectors'''
 
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- 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)
 
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- 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)
 
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- 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
 
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{|class="wikitable" style="text-align:center" border="1"
 
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|-
 
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!  Vectors !! Enzyme 1 !! Enzyme 2 !! Buffer !! Entire size !! Size of part1 !! Size of part2
 
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|-
 
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| ECE112|| XbaI || EcoRI || Fast Digest || 10156 || 6900 || 3295
 
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|-
 
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| ECE147|| EcoRI || HindIII || EcoRI || 5482 || 4913 || 600
 
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|-
 
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| ECE149|| SpeI || PstI || Fast Digest || 6192 || 4919 || 1300 
 
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|-
 
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| ECE150|| SpeI || HimdIII || Buffer 2 || 6166 || 5300 || 778 
 
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|-
 
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| ECE151|| SpeI || EcoRI || Fast Digest || 6166 || 4825 || 1300
 
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|-
 
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| ECE153|| SalI ||  || Buffer 3 || || ||
 
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|-
 
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| ECE162|| SalI ||  || Buffer 3 || 7600 || 6000 || 1600 
 
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|-
 
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| ECE165|| EcoRI || HindIII || EcoRI || 5952 || 5200 || 793 
 
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|-
 
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| ECE166|| EcoRI || HindIII || EcoRI || 7262 || 5100 || 2103
 
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|-
 
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| ECE171|| EcoRI || PstI || Fast Digest || 5444 || 3600 || 1826
 
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|-
 
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| ECE172|| HindIII ||  || Buffer 2 || 6462 || 3900 || 2540 
 
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|-
 
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| ECE176|| EcoRI || XbaI || Fast Digest || 866 || 4250 || 5128
 
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|}
 
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* '''Results'''
 
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* Lane1 : HyperladderI
 
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* Lane2 : ECE112
 
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* Lane3 : ECE147
 
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* Lane4 : ECE149
 
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* Lane5 : ECE150
 
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* Lane6 : ECE151
 
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* Lane7 : ECE162
 
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* Lane8 : ECE165
 
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* Lane9 : ECE166
 
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* Lane10 : ECE171
 
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* Lane11 : ECE172
 
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* Lane12 : HyperladderI
 
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[[Image:photoi.gif|300px|center]]
 
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- ECE112, ECE165, ECE166, ECE171 : right bands, ok
 
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- ECE172 : no band, not enough DNA
 
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- ECE147, ECE150, ECE162 : only one band, maybe not enough DNA
 
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- ECE149 : wrong size!
 
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- ECE151 : not sure, check again
 
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*'''Plates'''
 
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- Streak new plates with different strains of Bacillus : 1A1, IA751 and IA771
 
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Revision as of 07:58, 5 September 2008



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