"
Team:University College London/LabBook/Week6
From 2012.igem.org
Contents |
Tuesday 17.7.12
Aim -Testing the competency of the Reinvigorated Cell Line (W3110): After our original attempt to generate competency of the W3100 cell line was inadequate, we undertook a reinvigoration of the W3100 cell line (Expt 5.2). Here we aim to test the competency of the reinvigorated cell line using the same protocol as used in Expt 5.3, by evaluating their growth after transformation with a plasmid of a known high concentration (297ng/ul).
Method
(LOGO) Transformation Protocol 2
Step 1 – Thawing Cells: Use the reinvigorated W3100 cell line created in Week 5 (Expt 5.2)
Step 3 – Addition of BioBrick: To one 2ml eppendorf, add 1ul of pTop plasmid, and to another add nothing – this will be a control.
Step 7 – Adding Broth: SOC media was used, as it is preferred for this protocol.
Step 8 - Incubation: The table below indicates the Ampicillin concentration of the Agar gels.
| Samples | Volume Innoculated | Antibiotic in Gel (conc) | |
|---|---|---|---|
| Plasmid | pTOP | 36ul | Ampicillin (50ug/ml) |
| Control | Positive (No Plasmid) | 36ul | No Antibiotic |
| Negative (No Plasid) | 36ul | Ampicillin (50ug/ml) | |
Wednesday (18.7.12)
Aim - Results from Transformation
Result: Table below indicates there was no growth for our cells, but that the controls worked. Also included is an image of each plate.
| Samples | Growth/No Growth | |
|---|---|---|
| Plasmid | pTOP | No Growth |
| Control | Positive (No Plasmid) | Growth |
| Negative (No Plasid) | No Growth | |
Wednesday (18.7.12)
Aim - Testing the transformation protocols: In light of the problems with cell competency, we decided to test whether the choice of protocol was contributing to the poor transformation results – especially as it would require another week to set up another cell line. We used a known competent cell line, and tested its competency against both of our cell lines, for Transformation Protocol 1 vs Transformation Protocol 2. Each sample was plated on an Ampicillin positive Agar and incubated overnight.
(LOGO) Transformation - Protocol 1
(LOGO) Transformation - Protocol 2
Step ?: The table below describes the plates necessary for this investigation, each of which shoud carry Ampicillin antibiotic at a concentration of 50ug/ml.
| Plates | |
|---|---|
| Protocol 1 | W3100 – Original |
| W3100 – Reinvigorated | |
| W3100 – Known Competent | |
| Protocol 2 | W3100 – Original |
| W3100 – Reinvigorated | |
| W3100 – Known Competent | |
Thursday 19.7.12
Aim - Results from Transformation of pTop
Results: Table below indicates there was significant growth from the Original W3100 cells, but not from other cell lines.
| Plates | Colony Formation | |
|---|---|---|
| Protocol 1 | W3100 – Original | Yes |
| W3100 – Reinvigorated | No | |
| W3100 – Known Competent | No | |
| Protocol 2 | W3100 – Original | Yes |
| W3100 – Reinvigorated | No | |
| W3100 – Known Competent | No | |
Conclusion: The Original Cell line appears to be competent for both Protocols, while our reinvigorated cell line does not appear to be competent with either. The cells known to be competent showed no growth, for reasons that are unclear. For our Original W3100 cell line, there was greater colony formation with Protocol 1 than with Protocol 2, so we shall proceed with this protocol. It is likely that the failure of our transformations is due to the switch to protocol 2, and the use of too little BioBrick to transform our cells. The protocol recommends 1-2ul, but members around the lab generally use 5ul for transforming cells. We will take this approach and see if our transformations improve.
6.3
