Hi all, once again electroporation of Pichia proves troublesome. I am electroporating GS115 strain with a pPIC9K plasmid. Following three days of growth, both the MD and YPD plates contained pink colonies (see images). I was very careful in my preparation of the electrocompetent cells (all new reagents, everything performed in laminar flow hood). I also plated controls of pre-electroporation and post-electroporation cells on YPD and there does not appear to be any contamination on these plates.
Am I correct in thinking that this is simply just contamination of some sort? If anyone has any suggestions or alternative transformation protocols for me, I would greatly appreciate it!
Thanks,
Laura
Hi Laura,
You could try expanding a colony and isolating its DNA for sequencing (specific regions defining the species that you are using). You could try to compare the suspected colonies' sequence with the non-transfected cell DNA. Hope it helps!
Agree with Didem, good suggestion to sequence for identification, or use some other microbial identification technique.
You could also pick a colony and culture it in the absence of your selection marker and see if when you restreak it onto agar does the colour persist. This could indicate whether the colour is a result of your selection plasmid / gene of interest.
In practice though, it may be most time efficient (and least satisying) to simply try again using a fresh stock of P.P., have success and never know what happened.
Hi Laura, a good and quick assay if this is Pichia or a contamination is the following: Use your nose! Does it smell familiar / similar to the Pichias you have grown before. Or does it stink? In the latter case this is a contamination. Just start again, something has gone wrong and it takes you more time to find out what it was then just doing it again. However, when problems persist, it is required to follow them up.
Jü
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