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Team:SUSTC-Shenzhen/Notebook/Plasmid-purification-for-MU2-and-Struggles-II-for-the-first-time-construction-of-5X-UAS-mCherry-pX330-and-7X-UAS-mCherry-pX330
From 2014.igem.org
Notebook
Elements of the endeavor.
Contents |
Plasmid purification for M+U2 and Struggles II for the first-time construction of 5X-UAS -mCherry-pX330 & 7X -UAS-mCherry-pX330
2014/8/27
Plasmid purification (no endotoxin) for M+U2
Since we have confirmed that M+U2 is a successful one, we performed plasmid purification (no endotoxin) for it.
Method
Performed as protocol
Results
Concentration :130.5ng/ul
Struggles II for the first-time construction of 5X-UAS -mCherry-pX330 & 7X-UAS-mCherry-pX330
Till now, we still haven’t got one successful M+U+G monoclone. Thus, we then performed restriction digestion for the remaining undigested 6.5ul ligation reaction I, and transformed…
Materials
Enzymer: EcoRI
Buffer: Buffer2.1
The remaining undigested 6.5ul ligation reaction I
The usual things needed for transformation
Method
=Restriction digestion with EcoRI
Digestion system: [uint: ul]
| Ligation reaction I | EcoRI | Buffer | ddH2O | Total |
|---|---|---|---|---|
| 6.5 | 0.5 | 1 | 2 | 10 |
Incubate, 37C, 4h
Heat-inactivation
70C, 10min
Transformation
Performed as protocol
[10ul DNA to 100ul competent cell]
Results
For the newly digested-and-transformed group, there were no colonies growing out on the 5XUAS+mCherry-pX330 plate (It doesn’t matter, since we still got one successful M+U monoclone). And there was only one colony growing out on the 5XUAS+GAL4-mCherry-pX330 plate.
