Biomedical Engineering Reference
In-Depth Information
4 Add 250
μ
l of buffer P2 to each tube and mix.
5 Add 350
μ
l of buffer P3 to each tube and mix.
6 Spin tubes for 4 min at maximum speed.
7 Transfer the supernatant to new tubes.
8 Spin for 4min at the maximum speed to clear the supernatant.
9 Transfer the supernatant to new tubes.
10 Add 750
μ
l of isopropanol and keep at room temperature for 10min.
11 Collect the DNA by spinning the tubes for 10min at maximum speed.
12 Wash the DNA pellet once with 1 ml of 70% ethanol and air dry.
13 Resuspend all pellets in a total volume of 50
μ
l water (total from three tubes).
14 For electroporation use 1
μ
l of the BAC DNA.
PROTOCOL 10.2 Transformation of BAC into Recombinogenic
Bacterial Strain (DY380)
Equipment and reagents
•
Decay-wave ECM 630 electroporator (BTX - Genetronics)
•
DY380 cells (National Cancer Institute)
•
LB media with appropriate antibiotic resistance
•
15ml tubes
•
Spectrophotometer
•
Microcentrifuge at 4
◦
C
•
0.1 cm gap electroporation cuvette
•
1.5 ml Eppendorf tubes
•
LB-agar plates with appropriate antibiotic selection
32
◦
C incubator
•
•
Thermal cycler.
Method
1 Grow DY380 cells in 5ml of LB broth at 32
◦
C overnight with shaking.
a
2 On the next day, check cell growth by spectrophotometer reading (OD
600
=
1.2).
3 Collect the cells by centrifuging at 2000
g
for 5 min at 4
◦
C.
4 Resuspend the cell pellet in 1ml of ice-cold water and transfer to a 1.5ml Eppendorf
tube (on ice).
