"
Team:Groningen/Notebook/7 August 2009
From 2009.igem.org
(Difference between revisions)
(→Metal Accumulation) |
(→Metal Accumulation) |
||
| Line 43: | Line 43: | ||
**Use pET29a-SmtA to amplify SmtA | **Use pET29a-SmtA to amplify SmtA | ||
**Program: MT NIENKE (Left PCR machine) | **Program: MT NIENKE (Left PCR machine) | ||
| - | ***Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient | + | ***Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from 60-50°C (4steps), elongation time of 1:20. |
**Mix | **Mix | ||
::MM NH4 21uL | ::MM NH4 21uL | ||
Revision as of 11:15, 7 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
|
|---|
Wet
GVP Cluster
- → TODO isolate plasmids from overnight precultures
- → TODO perform a restriction on isolated plasmid (should result in 6000 and 3000bp fragments)
- → TODO plate on cultures for pure colony over the weekend
- → TODO cut pSB3K3 plasmid and GVP containing plasmid for ligation
- → TODO purify wanted fragments
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] with high, medium and low constitutive promoters and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 GVP] with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 20μL MQ and stored in the fridge
Concentration of Plasmids
| Plasmid | ng/μL | 260/280 | 260/230 | Control |
| GVP (biobrick) | 484.0 | ? | ? | ? |
Transporters
Metal Accumulation
- 2nd PCR to amplify SmtA and GST-SmtA
- Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA
- Use pET29a-SmtA to amplify SmtA
- Program: MT NIENKE (Left PCR machine)
- Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from 60-50°C (4steps), elongation time of 1:20.
- Mix
- MM NH4 21uL
- Primer fw [10mM] 1uL
- Primer rev [10mM] 1uL
- Vector 1uL
- HomeTaq pol 1uL
- Run program o/n
- Check on gel
- Transform E. coli with pGB68 (mymT)
- Use pGB68 from filter resuspended in 40uL MQ.
- As neg control use pSB1AC3 (incl ccdB), as pos control use pSB1AC3-low promoter.
- Mix 2-4uL of vector with 50uL chemically competent cells (prepared 31 aug)
- Transform using heat-shock @ 37°C
Vectors
Dry
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
