IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/05: Difference between revisions

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==Checking vectors==


- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153
==Starch plates==


- Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday)
Prepare some starch plates. We want to test our transformation but we can not manage to PCR Bacillus. So we are going to try to extract chromosomal DNA from Bacillus. We make these starch plates to test it with colonies which have positive control with amylase test.


- 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
- Prepare starch plates (you can find the protocol [[IGEM:Cambridge/2008/Turing_Pattern_Formation/Experiments/Bacillus_subtilis_transfomation#ECE153|here]])


*'''Results'''
- Plate 4 different plates :


* Lane3 : HyperladderI
* 2 colonies of IA75 (positive control) and 3 colonies of IA771 (negative control)
* Lane4 : ECE147
* 5 colonies of IA751 transformed with ECE153 from 29/08
* Lane5 : ECE149
* 3 colonies of IA751 transformed with ECE153 from 02/09 and 3 colonies from 03/09
* Lane6 : ECE150
* 5 colonies of IA751 transformed with ECE112 from 02/09
* 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


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Revision as of 03:58, 8 September 2008

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Starch plates

Prepare some starch plates. We want to test our transformation but we can not manage to PCR Bacillus. So we are going to try to extract chromosomal DNA from Bacillus. We make these starch plates to test it with colonies which have positive control with amylase test.

- Prepare starch plates (you can find the protocol here)

- Plate 4 different plates :

  • 2 colonies of IA75 (positive control) and 3 colonies of IA771 (negative control)
  • 5 colonies of IA751 transformed with ECE153 from 29/08
  • 3 colonies of IA751 transformed with ECE153 from 02/09 and 3 colonies from 03/09
  • 5 colonies of IA751 transformed with ECE112 from 02/09



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