Biology Reference
In-Depth Information
(a) IM-HS to select for the induction of
ADE2
and
HIS3.
(b) IM-MS to select for the induction of ADE2.
(c) IM-LS to select for the induction of HIS3.
(d) CM to check cell growth.
2. Incubate the plates at 30 °C. Monitor the emergence of colo-
nies during the next several days.
4
Notes
1. In order to confi rm that the fusion construct is in-frame, the
fusion junction can be sequenced using a standard T7 primer.
2. For best results, competent cells should be used for transfor-
mation immediately, although they can be stored on ice for a
few hours without signifi cant loss in effi ciency.
3. If expression of the bait has toxic effects, clone the bait in a
vector (such as pGBT9) with lower level of expression, or use
truncated versions of the protein to perform the screening.
4. Positive and negative controls must be included in all tests. For
GAL4 system vectors expressing Gal4 DNA-BD fused with
murine p53 (pGBKT7-53) and Gal4 AD fused with SV40
large T-antigen (pGADT7-T) are commonly used as positive
controls [
9
,
13
]. A negative control should also be performed
using pGBKT7-Lam (which encodes the Gal4 BD fused with
lamin C) and pGADT7-T.
Acknowledgments
We are grateful to Sharon White for critical reading the manuscript
and making many helpful suggestions. This research was supported
by a grant from the Spanish Ministerio de Ciencia y Tecnología
(AGL2010-22287-C02-02/AGR) and Fondo Europeo de
Desarrollo Regional (FEDER).
References
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