IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/19

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- Put them in the freezer at -80°C
- Put them in the freezer at -80°C
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==Dealing with R0040, R0062, S0168, I13522 for PA==
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* Plated R0040, R0062, S0168 received from MIT onto Amp100 plates and incubate at 37°C
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* Pciked 4 single colonies for each after colonies have grown and incubate overnight
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Check I13522 biobrick DNA
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* PCR biobrick DNA from 2007 wells
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* Run 0.8% EtBr E-gel
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* Lane 5: I13522
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* Lane 7: Hyperladder I
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Result:
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* Size of band is too small under 1000bp should be 2375bp instead
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* Transform TOP10 again but with I13522 from 2008 wells
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 +
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Contents

Results from the transformation with glycerol stocks

We used two different glycerol stocks, one from 07/08 and one from 13/08. We also had 2 different protocols (cf 18/08 page).

We tested our stocks on blank plates, cells are alive, the problem is to know if they are still competent or not.

  • Protocol 1 (with incubation time before adding DNA)

- Stock from 07/08 : nothing on the control and nothing on the transformation plate.

- Stock from 13/08 : nothing on the control and nothing on the transformation plate.

  • Protocol 2 (without incubation time before adding DNA)

- Stock from 07/08 : nothing on the control and nothing on the transformation plate.

- Stock from 13/08 : nothing on the control, and 1 colony on the transformed plate. We checked the fluorescence of bacteria into this colony (vector ECE166 with promoter and GFP). We have fluorescence, so our transformation is ok.

The result of this confirm that our stock of competent cells in glycerol is still competnet since we managed to transform one colony. The problem is certainly not the storage, but our protocol when we use frozen competent cells> We need to improve the efficiency of this protocol.

Test our Erythromycin stock

- We put some colonies on a Ery 0.5 plate. They grow, but in a very small amount, so our antibiotic seems to be fine.

- Cells in LB with antibiotic : no growth!

So our antibiotic is ok, either we did not transform our cells and that is why everything grew on Ery plates, either there is a problem in our protocol for Ery test.

Plasmid miniprep of ECE153, ECE166 and ECE171

- Plasmid miniprep LB stocks from yesterday

- Nanodrop

260/280 ng/μL
ECE 153 1.63 32.6
ECE 166 (1) 1.73 56.2
ECE 166 (2) 1.67 57.7
ECE 171 1.81 185.5

Transformation of IA771

We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171.

Spectrophotometer : blank made with medium A

Time (min) 0 20 40 80
OD650 0.1512 0.1498 0.1476 0.1535

There was a problem with our colonies. At t=80min, we inoculated again our tube of Medium A.

- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171

- Spectrophotometer : blank made with medium A

Time (min) 0 20 40 60 80 100
OD650 0.2393 0.2504 0.2663 0.2807 0.3184 0.3672

- Continue the protocol, add DNA (ECE153, 166, 171)

- Plate on antibiotic plates

  • New glycerol stocks

- 8 tubes : spin cells, throw away the supernatant, add 500μL of glycerol 60%

- 8 tubes : spin cells, keep only 100μL of medium B, resuspend cells and add 500μL of glycerol 60%

- Put them in the freezer at -80°C

Dealing with R0040, R0062, S0168, I13522 for PA

  • Plated R0040, R0062, S0168 received from MIT onto Amp100 plates and incubate at 37°C
  • Pciked 4 single colonies for each after colonies have grown and incubate overnight

Check I13522 biobrick DNA

  • PCR biobrick DNA from 2007 wells
  • Run 0.8% EtBr E-gel
  • Lane 5: I13522
  • Lane 7: Hyperladder I

Result:

  • Size of band is too small under 1000bp should be 2375bp instead
  • Transform TOP10 again but with I13522 from 2008 wells


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