Biomedical Engineering Reference
In-Depth Information
5 Centrifuge in a microcentrifuge at maximum speed for 15-20 s at 4
◦
C.
6 Place tubes on ice and aspirate the supernatant.
7 Repeat steps 3-5 two more times.
8 Resuspend the cell pellet in 50
μ
l of ice-cold water mixed with 1
μ
l of BAC DNA (from
Protocol 10.1).
9 Transfer to a chilled electroporation cuvette.
10 Electroporate once using the following conditions: 1.75 kV, 25
μ
F and 200 .The time
constant should be around 5-6 ms.
11 Add 1 ml of LB to the cuvette, transfer to an Eppendorf tube and incubate at 32
◦
C
for 1 h.
12 Spin down cells and spread the entire volume on one LB-agar plate containing the
appropriate antibiotic resistance. Incubate at 32
◦
Covernight.
b
Notes
a
It is important to always grow the recombineering strains at the restrictive temperature of
30-32
◦
C.
b
It is critical to screen the DY380 colonies by PCR to ensure that they contain both ends of
the BAC DNA or, preferably, to ensure that they contain both ends of the sequence that will be
retrieved from the BAC.
PROTOCOL 10.3
Preparation of PCR Product
Equipment and reagents
•
Thermal cycler
•
Dpn
I restriction enzyme (New England Biolabs)
•
High-fidelity
Taq
or
Pfu
enzyme and buffer (e.g. Stratagene)
•
37
◦
C water bath
Zymoclean gel purification kit (Zymo Research)
•
70% ethanol
•
Spectrophotometer.
•
Method
1 Design primers with 50 bp homology to the BAC sequence and approximately 20 bp
priming site on target cloning vector (pNAPP or pBSK; see Figure 10.2).
2 Dilute the cloning vector DNA in water to 0.1 ng/
μ
l (approximately 1 : 10 000 dilution
of mini prep DNA) and use 1
μ
l as a template for the PCR reaction.
