Biology Reference
In-Depth Information
Table 3
Supplements to be added to MM, CM and IMs media
Interaction media (IM)
Low
stringency
(IM-LS)
Medium
stringency
(IM-MS)
High
stringency
(IM-HS)
Minimal media
(MM)
Co-transformation
media (CM)
Amino acid
Lysine
+
+
+
+
+
Methionine
+
+
+
+
+
Uracil
+
+
+
+
+
Leucine
+
−
−
−
−
Tryptophan
a
+
−
−
−
−
Adenine
+
+
+
−
−
Histidine
+
+
−
+
−
3-AT
−
−
+
−
+
a
Stock must be protected from light and kept at 4 °C
sulfate (5 g/l), glucose (20 g/l) and a mixture of amino acids
to complement the yeast auxotrophies (Table
3
). Adjust the
pH to 6.5, then autoclave. If preparing solid MM, add agar
(20 g/l) and then autoclave it. Amino acid solutions must be
sterilized separately and then added before pouring plates.
YNB is a base medium for preparation of minimal and syn-
thetic defi ned yeast media that can purchase from different
companies (for example Becton, Dickinson, BD)
.
2.
Cotransformation medium
(CM). It is used to select for co-
transformants cells that contain both plasmids, the DNA-
binding domain (BD) and transcription activation domain
(AD) containing vectors. It has the same composition as MM
but it is not supplemented with leucine and tryptophan.
3.
Interaction media (IM).
They are employed to assess protein-
protein interactions. Three different IM media are defi ned to
control the stringency of the selection: IM-LS for low strin-
gency, IM-MS for medium stringency and IM-HS for high
stringency. The media have the same composition as CM, except
that adenine and/or histidine are absent and 3-AT (
3-Amino-
1,2,4-triazole
)
is added when histidine is not present.
4.
3-Amino-1,2,4-triazole
(3-AT; Sigma-Aldrich) is a competitive
inhibitor of the HIS3-gene product and may be used to titrate
the minimum level of
HIS3
expression required for growth on
histidine-defi cient media. A 2 M stock solution should be freshly
prepared by dissolving 3-AT in H
2
O and fi lter-sterilized.
