U34887, Genbank entry for pRSQ
YIp5
Vectorette PCR
Carl Friddle has developed a vectorette protocol for identifying the site of insertion. This can be found at http://genome-www.stanford.edu/group/botlab/protocols/vectorette.html. PCR
When pRSQ2-URA3 integrates into the transposon it creates an 11.7 kb insertion. This element is not cleaved by the following enzymes: AvrII, BglII, BspEI, EagI, MscI, NaeI, NheI, NruI, NotI, PmlI, SmaI, SnaBI, SpeI, SphI, XmaI. These enzymes can therefore be used to recover a large plasmid containing sequences both 5' and 3' to the transposon insertion. We have successfully 'moved' disruptions by this strategy. Note that this strategy does not move the allele as a lacZ-fusion. yIP5 can be used if this is required.
Antibiotics used:
| Kanamycin, Kan (Sigma K800) |
10 mg/ ml in water. Use at 40 ug/ml (Kan40) |
| Ampicillin, Amp (Sigma A9518) |
50 mg/ml in water. Use at 50 ug/ml (Amp50) |
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