IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/04: Difference between revisions
Yee Yen Tang (talk | contribs) (Autocreate 2008/08/04 Entry for IGEM:Cambridge/2008/Notebook/Bacillus) |
Yee Yen Tang (talk | contribs) |
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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 | |||
{|class="wikitable" style="text-align:center" border="1" | |||
|- | |||
! 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 | |||
[[Image:photoi.gif|300px|center]] | |||
- 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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Revision as of 04:59, 5 September 2008
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Wet Work
- 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
- 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
- Streak new plates with different strains of Bacillus : 1A1, IA751 and IA771 |