Hi All, I have cloned my gene of interest into pPIC9 vector following electroporation in GS115 strain. I used HIS4 gene for the selection and I got many colonies.Now, the colony should all be carrying the integrated pPIC9-Gene of interest.However, there is a possibility of getting false colony as well(I do not know how). Could anyone be able to explain the possibility of the false colony after electroporation?
Kindly screen for the transformants to confirm the integration of those genes in them before any other thing
You can get "satellite colonies" that are growing in a ring around resistant colonies and taking advantage of the low antibiotic environment.
Also, mutations always happen. Some of your bacteria can have resistance to the selective antibiotic even without the plasmid. Basically, mutations happen, even if the odds are 1 in a billion you will see it with bacteria.
You can also have ordinary lab mistakes. Using too low of a concentration of antibiotics, using plates that are old, contamination with a resistant bacteria, etc.
Good luck!
Not all colonies would carry your gene of interest.Some would be self ligation-depending on how you ve cloned yr gene.Is it a single digestion cloning that youve done-then the likelihood of getting self ligated vectors is more which will not amplify your gene of Interest
Please check these colonies by Colony PCR-using appropriate controls
Well, the pPIC9 has the origin of replication of bacteria only, not for the Yeast.In that case, how come a self ligated vector would give me false colony? It should only give me colony if it is integrated in to the AOX1 gene of GS115 strain(His4) and selective media does not have any amino acids. Now explain?
Dear Katie, Where does the antibody come from? You do not need to add antibody for selection of Pichia. I am working with Pichia pastoris not any bacteria. Either you did not read my post properly or I have huge gap in knowledge.
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