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Team:Groningen/Notebook/17 July 2009
From 2009.igem.org
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:::→{{todo}} Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made! | :::→{{todo}} Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made! | ||
:::→{{done}} Also plasmid from the o/n cultures will be purified and analysed by restriction analysis. | :::→{{done}} Also plasmid from the o/n cultures will be purified and analysed by restriction analysis. | ||
| - | * | + | *For plasmid isolation the Sigma Plasmid isolation kit was used (eluted in 50ul MQ). |
*DNA concentrations: | *DNA concentrations: | ||
| Line 34: | Line 34: | ||
|2.21 | |2.21 | ||
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| + | *Restriction analysis with SpeI and PstI to find insert length. | ||
| + | **Expected length with RFP insert ~1kb | ||
| + | **Expected length with gvp insert ~7kb | ||
| + | |||
| + | Restriction mixture for J23100+gvp (fragment sizes: 2096 (promoter + plasmid backbone), 887) | ||
| + | * 10 μL promoter plasmid (61.4 ng/μL, 96.1 ng/μL) | ||
| + | * 6μL MQ | ||
| + | * 2μL Fast digest buffer | ||
| + | * 1μL PstI fast digest enzyme | ||
| + | * 1μL SpeI fast digest enzyme | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 11:14, 17 July 2009
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Wet
GVP Cluster
Discussion:
All colonies (transformants vector + promoter ligated with gvp) and o/n culture became red, so probably RFP is still in there... What might be the problem? The vector with promoter self-ligated due to uncomplete digestion (done 15 July 2009) leading to the following fragments:
- vector+RFP linear
- vector
- RFP
When the upper fragment (~2kb) is contaminated with linear vector this would lead high efficiency self-ligation instead of ligating gvp with the emtpy vector.
- →TODO Therefore the restriction/purification/ligation will be redone and selection of non-red colonies will be made!
- →DONE Also plasmid from the o/n cultures will be purified and analysed by restriction analysis.
- For plasmid isolation the Sigma Plasmid isolation kit was used (eluted in 50ul MQ).
- DNA concentrations:
| Sample | ng/ul | 260/280 | 260/230 |
| gpv-promoter test (colony 9) | 82.5 | 2.02 | 2.21 |
| gpv-promoter test (colony 10) | 71.9 | 1.96 | 2.21 |
- Restriction analysis with SpeI and PstI to find insert length.
- Expected length with RFP insert ~1kb
- Expected length with gvp insert ~7kb
Restriction mixture for J23100+gvp (fragment sizes: 2096 (promoter + plasmid backbone), 887)
- 10 μL promoter plasmid (61.4 ng/μL, 96.1 ng/μL)
- 6μL MQ
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
Transporters
Metal Accumulation
Vectors
Dry
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