IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/05

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(Autocreate 2008/08/05 Entry for IGEM:Cambridge/2008/Notebook/Bacillus)
Current revision (06:57, 5 September 2008) (view source)
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==Entry title==
+
==Checking vectors==
-
* Insert your content here.
+
 +
- 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

Current revision



Main project page
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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


Very low bands : not enough DNA!!!

Transformation of Bacillus

  • Result of Nanodrop
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



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