IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/07/30: Difference between revisions

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<div id="about">
{{Cambridge08a}}
</div>
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__NOTOC__
{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:IgemCam.jpg|200px]]<span style="font-size:22px;"> Turing Pattern Formation</span>
|style="background-color: #444444"|[[Image:Signalling_button.gif|200px|center]]
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Wet Work==
==Wet Work==


* '''Transforming Bacillus Subtilis with medium A and medium A + tryptophan'''
* '''Checking our big stock of biobricks and PNZ plasmid'''
 
 
We did big culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.
 
- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks


Medium A
- Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation


{|class="wikitable" style="text-align:center" border="1"
{|class="wikitable" style="text-align:center" border="1"
|-
|-
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 73 !! 85 !! 95 !! 105
!  
! 260/280
! DNA concentration (ng/nl)
|-
| I746001 || 1.79 || 50.5
|-
| I746101 || 1.87 || 54.3
|-
|-
| OD650 || 0.1394 || 0.1370 || 0.1660 || 0.2304 || 0.2778 || 0.3138 || 0.3373 || 0.3583
| PNZ8901 || 1.84 || 87
|-
|-
|}
|}


For this medium, the curb stop to increase logarithmically at 100min.


- Digest


Medium A + tryptophan
{| class="wikitable"
 
|-
{|class="wikitable" style="text-align:center" border="1"
! For I746001 and I746101
! For PNZ8901 plasmid
|-
| 14μL of SDW
| 14μL of SDW
|-
| 2μL of 10X Fast Digest Buffer
| 2μL of Buffer 3 (Biolabs)
|-
|-
! Time (min) !! 0 !! 20 !! 40 !! 60 !! 73 !! 85 !! 95
| 2μL of DNA of biobricks
| 2μL of DNA of PNZ8901
|-
|-
| OD650 || 0.1493 || 0.1636 || 0.2080 || 0.2909 || 0.3512 || 0.4002 || 0.4165
| 1μL of EcoRI
| 1μL of PstI
|-
|-
| 1μL of SpeI
| 1μL of SalI
|}
|}


For this medium, the curb stop to increase logarithmically at 90min.


- Gel


- Incubate 90min at 37°C by shaking
* '''Results from this gel'''


- Prepare 7 tubes for medium A and 7 tubes for medium A + tryptophan : add 0.45mL of prewarmed medium B and 0.05mL of culture and incubate 90min at 37°C by shaking.
* Lane 9 : I746001
* Lane 10 : I746101
* Lane 11 : PNZ8901
* Lane 12 : HyperladderI


- Transform B.S. by adding DNA and incubate 30min at 37°C
[[Image:photob.gif|300px|center]]


We have one transformation with ppL82 (add 7.5μL of DNA), one with PNZ8901 (add 10μL of DNA) and one negative control without DNA.
Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.


- Plate 500μL on Cm plate (35μg/mL)
* '''New gel to check'''


- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901).


- For the remaining tubes, in one, add 60μL of glycerol and keep it in the freezer and keep the other one on the bench (for each medium)  
- Do single digest for PNZ8901 (one with PstI, one with SalI)


- Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)


* '''Checking our big stock of biobricks and PNZ plasmid'''
* '''Result from this second gel'''


[[Image:photoc.gif|300px|center]]


We didbig culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.
The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem.  With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks.  
 
- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks
 
- Digest
 
 
{| class="wikitable"
|-
! BGSC Accession
! Vector Name
! Features
! Vector Map
! Vector Sequence
|-
| ECE165
| vector name
| features
| map
| seq link
|-
| ECE166
| vector name
| features
| map
| seq link
|-
| ECE188
| vector name
| features
| map
| seq link
|-
| ECE189
| vector name
| features
| map
| seq link
|-
| ECE190
| vector name
| features
| map
| seq link
|}


Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.




Revision as of 04:35, 5 September 2008

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Wet Work

  • Checking our big stock of biobricks and PNZ plasmid


We did big culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.

- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks

- Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation

260/280 DNA concentration (ng/nl)
I746001 1.79 50.5
I746101 1.87 54.3
PNZ8901 1.84 87


- Digest

For I746001 and I746101 For PNZ8901 plasmid
14μL of SDW 14μL of SDW
2μL of 10X Fast Digest Buffer 2μL of Buffer 3 (Biolabs)
2μL of DNA of biobricks 2μL of DNA of PNZ8901
1μL of EcoRI 1μL of PstI
1μL of SpeI 1μL of SalI


- Gel

  • Results from this gel
  • Lane 9 : I746001
  • Lane 10 : I746101
  • Lane 11 : PNZ8901
  • Lane 12 : HyperladderI

Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.

  • New gel to check

- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901).

- Do single digest for PNZ8901 (one with PstI, one with SalI)

- Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)

  • Result from this second gel

The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem. With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks.

Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.



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