"
Team:BCCS-Bristol/Wetlab/K381001 Construction
From 2010.igem.org
(Difference between revisions)
m |
|||
| Line 38: | Line 38: | ||
* Only the commercial cells were successful and provided us with colonies | * Only the commercial cells were successful and provided us with colonies | ||
* Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added) | * Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added) | ||
| - | * | + | * Combined 3mL of cell culture from these overnights in 1.5mL eppendorf tube 4 times - total of 12mL combined cell culture. Performed a miniprep on the combined culture using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 200μL of solution containing [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] biobrick in high concentration. |
| + | * Once the DNA had arrived, the same procedure was followed to obtain large amounts of BBa_K216005. | ||
Revision as of 11:22, 17 September 2010
Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.
BBa_I13522 was taken from well 8A kitplate 2 of the 2010 distribution BBa_K216005 was requested from the parts registry
Construction of the biobrick can be broadly broken down into
- Generating larger quantities of both our component biobricks
- Digesting each of these biobricks
- Ligating together the desired parts to create a new biobrick
- Generating larger quantities of this biobrick, checking and sequencing
Generating large quantities of DNA:
- Whilst waiting for DNA from the parts registry we tried to obtain large amounts of the constitutive GFP biobrick
- We initially tried transforming MG1655s with DNA from the kit plate but were unsuccessful. Suspected the problem was down to cells not being competent enough and insufficient DNA in kit plate.
- Thus we tried transforming two further strains; XL1 Blues and commercial NovaBlues (see table for details)
| XL1-blue positive control | 100μL cells + 2μL known plasmid solution |
|---|---|
| XL1-blue negative control | 100μL cells (untransformed) |
| XL1-blue + 2009 biobrick | 100μL cells + 5μL 2009 biobrick |
| XL1-blue + 2010 biobrick | 100μL cells + 5μL 2010 biobrick |
| Nova blue + 2009 biobrick | 100μL cells + 5μL 2009 biobrick |
| Nova blue + 2010 biobrick | 100μL cells + 5μL 2010 biobrick |
- Only the commercial cells were successful and provided us with colonies
- Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
- Combined 3mL of cell culture from these overnights in 1.5mL eppendorf tube 4 times - total of 12mL combined cell culture. Performed a miniprep on the combined culture using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 200μL of solution containing [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] biobrick in high concentration.
- Once the DNA had arrived, the same procedure was followed to obtain large amounts of BBa_K216005.
