Hello netters,
I am now just learning about yeast so please be patient if my questions
seem dumb.
The woman who is teaching me is doing a two hybrid system to screen for
protein interactions. The yeast strain she uses is AH109 (-ade, -his,
-lac z). She explained these mutations are the reporter genes. If you
grow yeast that has been transformed with two plasmids (one a bait
plasmid and one a library protein plasmid) and grow on LT DO plates,
then that means the transformation worked. I understand that. She then
said the next step is a plasmid rescue (the library protein plasmid),
foll by KC8 transformation. Next you isolate the bacterial plasmid, do
a miniprep and "retransform" the yeast (that has the bait plasmid in it)
to see if there is a true interaction.
MY question is: why do you need to plate out these "retransformations"
on 4 different media plates (LT, ALT, HLT, AHLT). It seems to me that
if you have growth on AHLT that means that a) the yeast successfully
integrated the library protein AND turned on ade and trp. So why the
other plates?
I am so confused :(
K
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