IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/19: Difference between revisions

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|style="background-color: #EEE"|[[Image:IgemCam.jpg|200px]]<span style="font-size:22px;"> Turing Pattern Formation</span>
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We believe that the problem was in the gel-extraction between ligation and transformation. Most of our plasmid was probably lost in this step. Next time we will directly use the results from the ligation reaction to transform. Although many bands were seen on the previous gel of the ligation reaction, we will check our transformation growth by single colony PCR to confirm transformation of the plasmid with our correct insert.
We believe that the problem was in the gel-extraction between ligation and transformation. Most of our plasmid was probably lost in this step. Next time we will directly use the results from the ligation reaction to transform. Although many bands were seen on the previous gel of the ligation reaction, we will check our transformation growth by single colony PCR to confirm transformation of the plasmid with our correct insert.


==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
{|class="wikitable" style="text-align:center" border="1"
|-
! !! 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
{|class="wikitable" style="text-align:center" border="1"
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! Time (min) !! 0 !! 20 !! 40 !! 80
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| 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
{|class="wikitable" style="text-align:center" border="1"
|-
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 80 !! 100
|-
| OD650 || 0.2393 || 0.2504 || 0.2663 || 0.2807 || 0.3184 || 0.3672
|-
|}
[[Image:Courbe1_10839_image001.jpg]]
- 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
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==Lux parts==
==Lux parts==

Revision as of 08:46, 5 September 2008

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Results of AgrA and C transformation

Transformation has failed. No colonies were visible for the plates of Agr A or C. The Puc9 positive control grew. We believe that the problem was in the gel-extraction between ligation and transformation. Most of our plasmid was probably lost in this step. Next time we will directly use the results from the ligation reaction to transform. Although many bands were seen on the previous gel of the ligation reaction, we will check our transformation growth by single colony PCR to confirm transformation of the plasmid with our correct insert.


Lux parts

To make the Lux Receiver, we need 4 different parts ;

  • R0040, TetR repressible promoter
  • SO168, luxR + double terminator
  • R0062, promoter activated by luxR
  • JO4630 (GFP + double terminator)

All these parts have been transformed into E.coli. We want to test them. R004, R0062 and JO4630 have already been tested, it should be fine. We received from the MIT R0040, R0062 and S068 already transformed into E.coli, so we want to check these stocks (which are certainly fine) and use them. For JO4630, we want to double check our transformation.

- Plate on antibiotic plates and do LB stocks of single colony from the MIT stock (R0040, R0062 and S0168).

- Put on Kan plates 4 different colonies from J04630 (transformation Amp plate) and also incubate these colonies into LB


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