I am beginner in vector designing. My aim is to transform yeast for xdh and xi genes. so please provide me some references and protocols.
Dear Nilesh,
are you want to design cloning or expression vector ? i think if you have decided for expression then its very necessary to know that what features vector should have for cloning and expression in yeast.
a marker/reporter gene along with promoter+ ori site for yeast+ a inducible/regulatory promoter +MCS
you can freely download SNAPGENE viewer/ SNAP GENE software, it can be used for vector designing, again the marker/ reporter gene can be amplified along with promoter from other vectors by designing the cloning primers for respective regions, you can also amplify the MCS and ori site from some other vectors, in the same way promter can also be ammplified.
but pls take it in mind that all the amplified sequences you have to add finally, giving a circular plasmid, so that for each primer terminals you have to add specific restriction sites ( so the amplified sequences can be ligated in a proper way),
again the KOZAK consesus sequences are required for transcription of cloned gene under the regulation of promoter, thats why you have to add also these sequences after the promoter and before the MCS, the distance between promoter and initiation codon is very crucial for % expression of cloned gene, it should be 5- 10 bases, keep all these things in mind and start.
ALL the best
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