IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/05
From OpenWetWare
(Autocreate 2008/09/05 Entry for IGEM:Cambridge/2008/Notebook/Bacillus) |
(→Entry title) |
||
| Line 10: | Line 10: | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
| - | == | + | ==Checking vectors== |
| - | + | ||
| + | - Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153 | ||
| + | - Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday) | ||
| + | |||
| + | - Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172 | ||
| + | |||
| + | *'''Results''' | ||
| + | |||
| + | * Lane3 : HyperladderI | ||
| + | * Lane4 : ECE147 | ||
| + | * Lane5 : ECE149 | ||
| + | * Lane6 : ECE150 | ||
| + | * Lane7 : ECE151 | ||
| + | * Lane8 : ECE1162 | ||
| + | * Lane9 : ECE172 | ||
| + | * Lane10 : hyperladderI | ||
| + | |||
| + | |||
| + | [[Image:photoj.gif|300px|center]] | ||
| + | |||
| + | Very low bands : not enough DNA!!! | ||
| + | |||
| + | ==Transformation of Bacillus== | ||
| + | |||
| + | * Result of Nanodrop | ||
| + | |||
| + | {|class="wikitable" style="text-align:center" border="1" | ||
| + | |- | ||
| + | ! Vector !! 260/280 !! ng/μL | ||
| + | |- | ||
| + | | ECE112 || 1.75 || 64.6 | ||
| + | |- | ||
| + | | ECE166 || 172 || 138.6 | ||
| + | |} | ||
| + | |||
| + | - Prepare medium A with tryptophan, and medium B | ||
| + | |||
| + | - add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771) | ||
| + | |||
| + | - Check OD every 20min | ||
| + | |||
| + | - Incubate 90min | ||
| + | |||
| + | - Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes | ||
| + | |||
| + | - Incubate 90min | ||
| + | |||
| + | - Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!) | ||
| + | |||
| + | - Incubate 30min | ||
| + | |||
| + | - Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again | ||
| + | |||
| + | - Incubate 24hours | ||
| + | |||
| + | - A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes | ||
| + | |||
| + | |||
| + | - Transformation from glycerol stock from 30/07/2008 | ||
| + | |||
| + | - Spin glycerol stocks, pipette out glycerol | ||
| + | |||
| + | - Add 0.5mL of medium B, incubate for 1 hour | ||
| + | |||
| + | - Add 10μL of ECE112 (640ng) | ||
| + | |||
| + | - Incubate 2hours | ||
| + | |||
| + | - Plate 200μL, and 10min later, still 200μL | ||
| + | |||
| + | |||
| + | ==New stocks== | ||
| + | |||
| + | - Do glycerol stock of I746001 and I746101 (no sterile glycerol) | ||
| + | |||
| + | - Put IA751, IA771 in 10mL LB | ||
| + | |||
| + | - Put ECE 176 in 10mL LB + antibiotic | ||
| + | |||
| + | - Reinoculate the tube of LB from yesterday with ECE166 plate | ||
| + | |||
| + | - ECE176 replated onto Amp100 + Cm5 | ||
<!-- ## Do not edit below this line unless you know what you are doing. ## --> | <!-- ## Do not edit below this line unless you know what you are doing. ## --> | ||
Revision as of 07:52, 5 September 2008
 |  Main project page Previous entry Next entry
| |||||||||
Checking vectors- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153 - Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday) - Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172
 Very low bands : not enough DNA!!! Transformation of Bacillus
- Prepare medium A with tryptophan, and medium B - add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771) - Check OD every 20min - Incubate 90min - Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes - Incubate 90min - Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!) - Incubate 30min - Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again - Incubate 24hours - A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes
- Spin glycerol stocks, pipette out glycerol - Add 0.5mL of medium B, incubate for 1 hour - Add 10μL of ECE112 (640ng) - Incubate 2hours - Plate 200μL, and 10min later, still 200μL
New stocks- Do glycerol stock of I746001 and I746101 (no sterile glycerol) - Put IA751, IA771 in 10mL LB - Put ECE 176 in 10mL LB + antibiotic - Reinoculate the tube of LB from yesterday with ECE166 plate - ECE176 replated onto Amp100 + Cm5 | ||||||||||
