Biology Reference
In-Depth Information
2. Inoculate one colony into 50 ml YPDA medium in a 250 ml
sterile fl ask. Incubate at 30 °C with shaking at 250 rpm
over-night.
3. Transfer an appropriate volume of the over-night culture to
300 ml of YPDA in a 500 ml fl ask to reach OD
600
of 0.2.
Incubate with shaking at 250 rpm until the OD
600
reaches
0.8-1 (6-8 h).
4. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Discard the supernatant and resuspend the pellet in
150 ml of sterile deionized water.
5. Repeat
step 4
.
6. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Discard the supernatant.
7. Add the solutions below to the cell pellet in the following order:
50 % PEG 4,000
14.4 ml
1 M Lithium acetate
2.1 ml
Salmon sperm-DNA (2 mg/ml)
1.5 ml
DNA from the bait-plasmid (10-20
μ
g)
X
μ
l
DNA from the library (5-10
μ
g)
Y
μ
l
Complete with sterile deionized water up to a fi nal volume of
3 ml.
The volumes can be scaled down or up to reach the
required number of transformants. For best results, competent
cells should be used for transformation immediately, although
they can be stored on ice for a few hours after the washes, with-
out signifi cant loss in effi ciency.
8. Mix vigorously and incubate at 30 °C for 30 min mixing by
inversion every 5 min to homogenize the temperature. It is
important to resuspend the cells completely before starting the
incubation.
9. Transfer the tubes to rack placed in a preheated 42 °C circulat-
ing water bath. Store the tubes in the rack for 30 min mixing
by inversion every 5 min to homogenize the temperature.
10. Centrifuge the cells at 3,000 ×
g
for 4 min at room tempera-
ture. Gently resuspend the cells in 30-40 ml of sterile deionized
water.
11. Remove 100
l from the transformation mixes and prepare
dilutions (10
−1
, 10
−2
, and 10
−3
) in sterile deionized water. Plate
100
μ
l of each dilution onto 10 cm diameter CM plates and
incubate at 30 °C. This will allow the total number of co-
transformants to be calculated.
μ
