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Team:Groningen/Notebook/7 August 2009
From 2009.igem.org
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The PCRs done yesterday (PCR 1 and2) were run on gel: | The PCRs done yesterday (PCR 1 and2) were run on gel: | ||
| - | [Image:F102471_2009-08-07_10hr_33min_LigationCheckandPCR12_noted.jpg] | + | [Image:F102471_2009-08-07_10hr_33min_LigationCheckandPCR12_noted.jpg|] |
===Metal Accumulation=== | ===Metal Accumulation=== | ||
Revision as of 12:19, 7 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → DONE isolate plasmids from overnight precultures
- → DONE 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 |
| pSB3K3-H | 25.0 | ? | ? | ? |
| pSB3K3-M | 32.6 | ? | ? | ? |
| pSB3K3-L | 33.9 | ? | ? | ? |
| GVP (biobrick) | 484.0 | ? | ? | ? |
| pSB1AC3-H-GVP-amp | 106.6 | 2.00 | 2.44 | ? |
| pSB1AC3-M-GVP-amp | 165.2 | 1.92 | 2.40 | ? |
| pSB1AC3-L-GVP-amp | 155.0 | 1.95 | 2.44 | ? |
| pSB1AC3-H-GVP-amp-conc. | 116.3 | 1.96 | 2.43 | ? |
| pSB1AC3-M-GVP-amp-conc. | 108.7 | 1.95 | 2.46 | ? |
| pSB1AC3-L-GVP-amp-conc. | 302.8 | 1.88 | 2.45 | ? |
| pSB1AC3-H-GVP-amp/chl-conc. | 257.1 | 1.92 | 2.41 | ? |
| pSB1AC3-M-GVP-amp/chl-conc. | 224.7 | 1.94 | 2.37 | ? |
| pSB1AC3-L-GVP-amp/chl-conc. | 166.1 | 1.95 | 2.44 | ? |
| pSB1AC3-L-GVP-amp/chl-oud | 223.9 | 1.91 | 2.31 | ? |
Transporters
GlpF The PCRs done yesterday (PCR 1 and2) were run on gel: [Image:F102471_2009-08-07_10hr_33min_LigationCheckandPCR12_noted.jpg|]
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
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