Team:University College London/LabBook/Week3

From 2012.igem.org

(Difference between revisions)
(Created page with "{{:Team:University_College_London/templates/headimg|coverpicture=images/a/a1/Ucl2012-labbook-title.png}}<html><script type="text/javascript" src="https://2012.igem.org/wiki/index....")
(→Wednesday (20.6.12))
 
(15 intermediate revisions not shown)
Line 2: Line 2:
</html>{{:Team:University_College_London/templates/labbookmenu}}<html>
</html>{{:Team:University_College_London/templates/labbookmenu}}<html>
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" />
<img src="https://static.igem.org/mediawiki/2012/d/d4/Ucl2012-labbook-monfri.png" />
-
<img src="https://static.igem.org/mediawiki/2012/1/13/Ucl2012-labbook-graph7-1.png" />
+
<img id="3-1" src="https://static.igem.org/mediawiki/2012/b/b4/Ucl2012-labbook-graph3-1.png" />
<div class="experimentContent"></html>
<div class="experimentContent"></html>
-
==Monday 23.7.12 ==
+
==Monday (18.6.12)==
-
'''Aim - Transformation of TetR BBa_C0040 BioBrick'''
+
Aim – Day 1 of Generating Competent Cells: In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.
-
(LOGO) Transformation Protocol 1
+
In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.
-
'''Step 1 –''' Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1)
+
'''Method'''
-
'''Step 3 –''' Addition of BioBrick: To a  2ml eppendorf, add 1ul of the following BioBricks. Note: we have changed the protocol for our positive control. Previously it contained no BioBrick, but it has been recommended to us that we transform our positive control such that there is one for each BioBrick – this will tell us if the BioBrick has in any way affected cell viability. This will be used from this point onwards. Include an extra tube as a negative control, with no BioBrick added
+
<html><div class="protocol protocol-Competency">Day 1 of Generating Competent Cells</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Competency}}<html></div></html>
-
{| class="wikitable"
 
-
|-
 
-
! colspan="2" |  Samples !! Function !! Module
 
-
|-
 
-
| BioBrick || BBa_C0040 ||Tetracycline Repressor || Buoyancy
 
-
|-
 
-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040) ||  ||
 
-
|-
 
-
| Negative (No Biobrick) ||  ||
 
-
|-
 
-
|}
 
-
'''Step 9 -''' Plating samples on Agar Plates: The table below indicates the chosen inoculation volume (two for each BioBrick) and the correct gel antibiotic concentration for all samples.
+
== Tuesday (19.6.12) ==
-
{| class="wikitable"
+
'''Aim – Picking Colonies:''' Today we check the colony formation from yesterday’s culture, and pick a colony for inoculation
-
|-
+
-
! colspan="2" |  Samples !! Volume Inoculated !! Antibiotic in Gel (ug/ml)
+
-
|-
+
-
| rowspan="2" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || rowspan="2" | Ampicillin(50ug/ml)
+
-
|-
+
-
| 90ul
+
-
|-
+
-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || No Antibiotic
+
-
|-
+
-
| Negative (No BioBrick)|| 36ul ||  2x Ampicillin(50ug/ml)
+
-
|-
+
-
|}
+
 +
'''Method'''
-
== Tuesday 24.7.12  ==
+
<html><div class="protocol protocol-Competency">Day 2 of Generating Competent Cells</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Competency2}}<html></div></html>
-
'''Aim - Results of Transformation'''
 
-
'''Result:''' The table below indicates that there was growth for this transformation.  
+
== Wednesday (20.6.12) ==
-
{| class="wikitable"
 
-
|-
 
-
! colspan="2" |  Samples !! Volume Inoculated !! Colony Formation
 
-
|-
 
-
| rowspan="2" |BioBrick ||rowspan="2" | BBa_C0040  || 10ul || Yes
 
-
|-
 
-
| 90ul || Yes
 
-
|-
 
-
| rowspan="2" | Control || Positive (Contains BioBrick BBa_C0040)|| 36ul || Yes
 
-
|-
 
-
| Negative (No BioBrick)|| 36ul || No
 
-
|-
 
-
|}
 
 +
'''Aim – Permeabilisation:''' Incubating Cells in the presences of CaCl<sub>2</sub> will prepare the cell wall, such that it becomes permeable to DNA.
-
<html></div>
+
'''Method'''
-
<div class="experiment"></div>
+
-
<img src="https://static.igem.org/mediawiki/2012/2/22/Ucl2012-labbook-graph7-2.png" /><div class="experimentContent">
+
-
</html>
+
-
== Hello World ==
+
<html><div class="protocol protocol-Competency">Day 3 of Generating Competent Cells</div><div class="protocolContent"></html>{{:Team:University_College_London/Protocols/Competency3}}<html></div></html>
-
bla bla bla
+
 
-
<html>
+
 
-
</div><div class="experiment"></div></html>
+
 
 +
<html></div>
 +
<div class="experiment"></div></html>
{{:Team:University_College_London/templates/foot}}
{{:Team:University_College_London/templates/foot}}

Latest revision as of 03:45, 27 September 2012

Monday (18.6.12)

Aim – Day 1 of Generating Competent Cells: In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.

In order to culture our BioBricks in a cell line, we must first enable our cells to uptake plasmid. Today we will be starting the first day of the protocol, whereby we streak the cells on a minimal agar.

Method

Day 1 of Generating Competent Cells


Step 1 – Setting up Plates: Pour several plates with minimal agar.

Step 2 – Streaking: Streak the chosen cell line onto the minimal agar plate.

Step 3 – Incubation: Incubate at 37oC overnight



Tuesday (19.6.12)

Aim – Picking Colonies: Today we check the colony formation from yesterday’s culture, and pick a colony for inoculation

Method

Day 2 of Generating Competent Cells

Step 1 -Creating culture media: In a sterile environment, set up a falcons, with 5mls of Lysogeny Broth and 100ul 1M MgS04.

Step 2 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop, scoop up a colony onto the tip. Deposit in the falcon tube

Step 3 - Cell culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.


Wednesday (20.6.12)

Aim – Permeabilisation: Incubating Cells in the presences of CaCl2 will prepare the cell wall, such that it becomes permeable to DNA.

Method

Day 3 of Generating Competent Cells

Step 1 – Innoculation: Inoculate 100mls of Lysogeny Broth in pre-warmed conical with 1ml of the overnight culture from Day 2

Step 2 – Incubation: Incubate for two hours in a 37oC shaker until the cells are at the early log phase of the growth curve. Measure absorbance on a spectrometer until A6000 is approximately 0.3.

Step 3 – Incubation: Transfer to a chilled sterile 50ml Falcon tube and incubate on ice for 10 minutes Step 4 – Centrifuge:

-Time: 5 mins -G – 3300 -Temperature: 180C

Step 5 – Incubate: Resus in 10ml of ice cold 0.1M CaCl2 in 15% glycerol and incubate on ice for 30 minutes.

Step 6 – Centrifuge: -Time: 5 mins -G – 3300 -Temperature: 180C

Step 7 – Transfer: Resus in 1ml of ice cold 0.1M CaCl2 in 15% glycerol. Transfer 100ul aliquots to pre-chilled, pre-labelled eppendorf tubes. Store at -70oC

Step 8 – Centrifuge -Time: 5 mins -G – 3300 -Temperature: 180C

Step 9 - Storage: Store cells at -80oC




Retrieved from "http://2012.igem.org/Team:University_College_London/LabBook/Week3"