Team:University College London/LabBook/Week5

From 2012.igem.org

(Difference between revisions)
(→Tuesday (10.7.12))
(→Monday (9.7.12))
Line 50: Line 50:
| rowspan="16" |BioBrick ||rowspan="2" | BBa_ K123003 || 10ul || rowspan="14" | Ampicillin(50ug/ml)
| rowspan="16" |BioBrick ||rowspan="2" | BBa_ K123003 || 10ul || rowspan="14" | Ampicillin(50ug/ml)
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_ K123002 || 10ul
+
| rowspan="2" | BBa_ K123002 || 20ul
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_B0034 || 10ul
+
| rowspan="2" | BBa_B0034 || 20ul
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_J23100 || 10ul
+
| rowspan="2" | BBa_J23100 || 20ul
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_J23107 || 10ul
+
| rowspan="2" | BBa_J23107 || 20ul
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_R0040 || 10ul
+
| rowspan="2" | BBa_R0040 || 20ul
|-
|-
-
| 90ul
+
| 200ul
|-
|-
-
| rowspan="2" | BBa_J23106  || 10ul
+
| rowspan="2" | BBa_J23106  ||20ul
|-
|-
-
| 90ul
+
| 200ul 
|-
|-
-
| rowspan="2" | BBa_ K398108 || 10ul || rowspan="2" | Chloramphenicol (25ug/ml)
+
| rowspan="2" | BBa_ K398108 || 20ul || rowspan="2" | Chloramphenicol (25ug/ml)
|-
|-
-
| 90ul
+
| 200ul
|-
|-
| rowspan="2" | Control || Positive (No BioBrick)|| 36ul || No Antibiotic
| rowspan="2" | Control || Positive (No BioBrick)|| 36ul || No Antibiotic

Revision as of 12:21, 1 August 2012

Contents

Monday (9.7.12)

Aim: Transformation of Key BioBricks

Method

(LOGO) Transformation Protocol 2

Step 1 – Thawing Cells: Use W3100 cell line created in Week 2 (Expt 2.1) Step 3 – Addition of BioBrick: To each 2ml eppendorf, add 1ul of the following BioBricks. Include an extra tube as a control, with no BioBrick added.

Function Module
BioBrick BBa_K123003 Oestrogen Receptor Detection
BBa_K123002 Oestrogen Response Element Detection
BBa_K398108 Salt Tolerance Cluster Salt Tolerance
BBa_J23100 Constitutive Promoter
BBa_J23107 Constitutive Promoter
BBa_R0040 TetR Repressible Promoter Buoyancy
BBa_J23106 Constitutive Promoter
BBa_B0034 Ribosome Binding Site (RBS) All
Control (No BioBricks)

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.



Samples Volume Inoculated Antibiotic in Gel (ug/ml)
BioBrick BBa_ K123003 10ul Ampicillin(50ug/ml)
200ul
BBa_ K123002 20ul
200ul
BBa_B0034 20ul
200ul
BBa_J23100 20ul
200ul
BBa_J23107 20ul
200ul
BBa_R0040 20ul
200ul
BBa_J23106 20ul
200ul
BBa_ K398108 20ul Chloramphenicol (25ug/ml)
200ul
Control Positive (No BioBrick) 36ul No Antibiotic
Negative (No BioBrick) 36ul 2x Ampicillin(50ug/ml)

1x Chloramphenicol (25ug/ml)

Tuesday (10.7.12)

Aim - Check results from Transformation

Results: The table below indicates whether or not there was growth on each plate. Included is an image demonstrating the growth noted for BBa_K123003, BBa_K398108, and the Positive Control

Samples Volume Inoculated Growth/No Growth
BioBrick BBa_ K123003 20ul Growth
200ul Growth
BBa_ K123002 20ul No Growth
200ul No Growth
BBa_B0034 20ul No Growth
200ul No Growth
BBa_J23100 20ul No Growth
200ul No Growth
BBa_J23107 10ul No Growth
2000ul No Growth
BBa_R0040 20ul No Growth
200ul No Growth
BBa_J23106 20ul No Growth
200ul No Growth
BBa_ K398108 20ul No Growth
200ul Growth
Control Positive (No BioBrick) 36ul Growth
Negative (No BioBrick) 36ul No Growth

Conclusion: Only BBa_K398108 and BBa_K123003 had successfully transformed. This is a serious setback, which suggests our cell competency is not as high as we first anticipated. Method

(LOGO) Picking Colonies

Step 2 - Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic used for the BioBrick.


4.1

5.3

5.2

Retrieved from "http://2012.igem.org/Team:University_College_London/LabBook/Week5"