Biomedical Engineering Reference
In-Depth Information
•
DNA purification kit
•
Shaking incubator
•
Heat block
Yeast host strains: PJ69-4A (MATa) for baits or PJ69-4
α
(MAT
α
) for preys.
e
•
YPAD full rich media for growth of untransfected host strain. Containing: 10 g/l yeast
extract, 100
μ
g/l adenine hemisulfate, 20 g/l peptone and 20 g/l
D
-glucose.
This media is
only used when no selection is required
.
•
•
SD media: 6.7 g/l yeast nitrogen base without amino acids, 20 g/l
D
-glucose and 20 g/l
agar. This provides a minimal media which can be supplemented in accordance with
required selection conditions. When using the PJ69-4A/
α
strains in combination with
pGBAD-B, pGBAE-B, pACTBD-B or pACTBE-B vectors for gap repair reactions, low adenine
conditions are required. Therefore, SD media should be supplemented with arginine and
methionine (20
μ
g/ml), isoleucine and lysine (30
μ
g/ml), phenylalanine (50
μ
g/ml),
valine 150 and 20
μ
g/ml adenine hemisulfate (as opposed to the standard 100
μ
g/ml
that would normally be used in non-selective media). This low level of adenine is
essential for the development of pink/red colonies, which indicate the presence of
vectors with in-frame inserts. In addition, leucine should be added at 100
μ
g/ml, while
uracil, tryptophan,
f
and histidine should be added at 20
μ
g/ml, as required for
appropriate selection of bait or prey plasmids. Yeast containing bait vectors (pGBAD-B or
pGBAE-B) contain the TRP1 gene and, therefore, will grow on media lacking tryptophan,
while prey vectors (pACTBD-B or pACTBE-B) contain the LEU2 gene, which facilitates
growth on media lacking leucine.
To prepare
Bam
HI linearized plasmid DNA: digest 2
μ
g of plasmid DNA with 20 U of
Bam
HI in 1
×
digestion buffer (NEB buffer 3 with 100
μ
g/ml BSA) for 1 h at 37
◦
C.
Following digestion, clean up DNA using a QIAquick PCR purification kit and adjust final
concentration to 20 ng/
μ
l.
•
Method
1 Inoculate 2 ml of YPAD broth with fresh yeast PJ69-4A (MATa) for baits or PJ69-4
α
(MAT
α
) for preys and grow overnight at 30
◦
C with shaking (
∼
220 rpm).
2 Next day add an additional 8ml of fresh YPAD broth and continue to incubate at 30
◦
C
with shaking for 5 h.
3 Harvest yeast by centrifugation for 5min at 2300 rpm (
∼
700
g
).
g
4 Resuspend yeast in 5ml of 100m
M
LiOAc and transfer 1.5 ml of the suspension to a
microcentrifuge tube. Pellet the cells as above.
5 Wash cells again in 100m
M
LiOAc and harvest them by brief centrifugation in a
microfuge at 2300 rpm (
∼
700
g
).
6 After final wash, remove supernatant and add 320
μ
l of the following master mix:
2.3ml 50% (w/v), PEG 3350, 350
μ
l1
M
LiOAc, 450
μ
l sterile distilled water, 90
μ
lof
10.5mg/ml heat-denatured salmon testis DNA as a carrier and 10
μ
lof20ng/
μ
lof
Bam
HI linearized plasmid DNA. This provides sufficient master mix for 10 reactions.
7 Mix well before transferring 32
μ
l into individual PCR tubes.
