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Team:Groningen/Notebook/6 August 2009
From 2009.igem.org
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[[image:F102471_2009-08-06_15hr_36min_metal_sensitive_prom_+_GVP_(2).JPG]] | [[image:F102471_2009-08-06_15hr_36min_metal_sensitive_prom_+_GVP_(2).JPG]] | ||
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| + | |1 1kb ladder | ||
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| + | |9 1kb ladder | ||
| + | |18 1kb ladder | ||
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===Transporters=== | ===Transporters=== | ||
Revision as of 15:18, 6 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
Ideas to improve ligation
- Blunt end ligation of vector with PCR fragment, use Klenow polymerase to blunt end the vector.
- Dephophorylation of the vector via CAP.
- Do not (preferably) clean up PCR product from gel, but use ~1uL for checking on gel and clean the rest with a PCR cleanup kit.
- Use conventional restriction enzymes for digestion of the insert and vector, as these can be heat-inactivated.
- Increase the amount of insert from 1:3 to 1:6 or 1:10.
- Digest insert with XbaI and SpeI, these give compatible overhangs but these sites are closer to the gene (and have longer spacer).
GVP Cluster
Over Night Cultures
Over night cultures in 5mL LB-amp50 or LB-amp25/-chloramph100 medium were prepared from the following colonies:
- → E.coli TOP10 pSB1AC3-high const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-high const. promoter - GVP (amp./chl)
- → E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp./chl)
- → E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp./chl)
- → E.coli TOP10 (oud) pSB1AC3-med. const. promoter - GVP (amp./chl)
and put in the 37°C waterbath at 200 rpm.
- → Note: The concentration of Chloramphe. antibiotics can be lower because crystalisation had occured during storage, vortexing did not dissolve the crystals completely!
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] with high, medium and low constitutive promoters and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016] 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
Results: metal sensitive promoters in front of GVP
If the construct is correct one band at ~8kb should be visible. If the construct is incorrect one band at ~8kb and another at ~1.5kb should be visible.
.JPG)
| 1 1kb ladder | 10 1kb ladder |
| 2 | 11 |
| 3 | 12 |
| 4 | 13 |
| 5 | 14 |
| 6 | 15 |
| 7 | 16 |
| 8 | 17 |
| 9 1kb ladder | 18 1kb ladder |
Transporters
Metal Accumulation
- Check PCR on SmtA and GST-SmtA on gel
- Run 20uL (+ 1:6 dye) on 1% gel
- Contents:
- 2=1kb marker
- 4=GST-SmtA PCR product 1 --> expected size: 1400bp
- 6=SmtA PCR product 1 -->expected size: 660bp
- 8=GST-SmtA PCR product SmtA -->expected size: 660bp
- No bands of expected size were found.. So PCR will be redone tomorrow.
Vectors
Dry
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