I'm actually trying to transform two plasmids into yeast. 1. pRS416 with URA3 gene, 2.pRS413 with His3 gene. At first, i have transformed pRS416 into yeast that worked (got colonies on -URA plate). Then i have used these colonies, grew them using -URA media and tried to transform intact/linearized pRS413 using the same procedure. And plated them on -URA-His plates. Now my cells had (probably!) intact pRS416 and intact/linearized pRS413. After 3 days, I have observed colonies with intactpRS416+ intact pRS413 but i didn't see any colony on intact pRS416+linearized pRS 413.
PS: we know that plasmid pRS416 can produce a protein that will repair linearized pRS413 to make it intact again.
Amounts: plasmid=50ng, SS-DNA=100ug.
Anyone has any suggestion to transform linearized pRS413 into yeast cells? Thanks in advance!
Hi there,
As you actually try to select a double event (transformation plus self ligation) you should first plate the transformants on non selective media for the second plasmid (-ura) for a couple of days and then replica plate onto selective media (-ura-his). Furthermore you should engage more linear plasmid (µg) in the transformation in order to get more transformants in order to increase the probability to get the self ligation event. The problematic is quite similar to transformation with linear DNA which has to insert into the genome by double recombination.
Thanks a bunch Dr. Dominique Liger! Today I have started with 250ng of plasmid instead of 5ng. Next time I’ll try to grow them on -Ura first and then on -Ura-His. I’ll let you know if that work!
@Dr Faris AbachI: we have solved our problem by stably integrating our expected gene into the yeast genome. So now we need to transform only one plasmid instead of two (as the expression of this integrated gene will repair the linearized plasmid). NB. We have used 250ng of linearized plasmid but we saw that more DNA will produce more colony!!
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