Biomedical Engineering Reference
In-Depth Information
3 Amplify the vector by high-fidelity PCR in a 50
μ
l reaction for 40 cycles (see DNA
polymerase manufacturer's instructions).
4Add1
μ
lof
Dpn
I restriction enzyme directly to the final PCR product and incubate at
37
◦
C for 1 h to digest the template DNA.
5 Gel-purify the PCR product using the Zymoclean gel purification kit. Perform an extra
70% ethanol wash to remove all salts prior to elution.
6EluteDNAin20
μ
l water and measure the absorption at 260 nm using a
spectrophotometer in order to calculate DNA concentration.
PROTOCOL 10.4
Retrieval of Sequence of Interest from BAC DNA
Equipment and reagents
•
LB media with appropriate selection
•
32
◦
C incubator
•
Spectrophotometer
•
42
◦
C water bath
•
Autoclaved 250ml flasks
•
15ml tubes
•
Centrifuge at 4
◦
C
•
LB-agar plates with appropriate antibiotic
•
0.1 cm gap electroporation cuvette
•
Spectrophotometer
•
Decay-wave ECM 630 electroporator (BTX - Genetronics).
Method
1 Grow DY380 cells containing the BAC in 5ml of LB broth with appropriate antibiotic at
32
◦
C overnight with shaking (220 rpm).
2 On the following day, transfer 1 ml of the overnight culture (OD
600
=
1.2) to a 250ml
flask containing 20ml of LB with antibiotic. Incubate for 2 h at 32
◦
C with shaking
(OD
600
=
0.5).
3 Transfer 10ml of the cells to a new 250ml flask and shake in a 42
◦
C water bath for
15min.
4 Put both flasks (42
◦
C treated and control) into wet ice and swirl to make sure that the
temperature drops as quickly as possible. Leave on wet ice for 5 min.
5 Transfer the cells to cold 15 ml centrifuge tubes and spin at 2000
g
for 5 min at 4
◦
C.
6 Resuspend the cells in 1ml of ice-cold water and transfer to a 1.5 ml Eppendorf tube
on ice.
