IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/05: Difference between revisions
Yee Yen Tang (talk | contribs) (Autocreate 2008/08/05 Entry for IGEM:Cambridge/2008/Notebook/Bacillus) |
Yee Yen Tang (talk | contribs) |
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== | ==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 | |||
Revision as of 04:57, 5 September 2008
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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
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