Elucidating Gene Function through Use of Genetically Engineered Mice - Genomics: Essential Methods - page 231

• Phenol chloroform
• Chloroform
• 70% ethanol
• Microcentrifuge
•

Spectrophotometer.

Method

1 Purify targeting vector plasmid DNA using the Qiagen endotoxin-free plasmid maxi kit
in order to obtain a high concentration and purity of DNA.
2 Linearize 80 μ g of targeting vector (in order to obtain at least 25 μ g after subsequent
purification) for 6-8 h in a 37 ◦ C water bath with an appropriate restriction enzyme
that has a unique cut site on one end of a homology arm; for example, between the TK
cassette and a homology arm. Use 25 μ l of enzyme and suitable buffers in a total
volume of 400 μ l.
3 Before purification, run 2 μ l of digested DNA on a 1% agarose gel to verify digestion
efficiency. If not completely digested, add more enzyme (total enzyme can be
40 μ l).
4 Add an equal volume of phenol chloroform, vortex and then spin for 3 min at maximum
speed in a bench-top centrifuge.
5 Draw off aqueous (top) phase (contains DNA) and add to it an equal volume of
chloroform (to remove phenol). Vortex and spin down for 3 min at maximum speed.
6 Draw off aqueous phase (contains DNA), and add to it 1/10 volume of 3 M sodium
acetate plus two volumes of 100% ethanol. Vortex and freeze at − 80 ◦ Cfor
15min.
7 Spin for 10min at 4 ◦ C at maximum speed to pellet the DNA.
8 Suck off the ethanol and wash the pellet with 150 μ l of 70% ethanol.
9 Spin for 3 min at maximum speed.
10 Draw off ethanol and air-dry the pellet for 15min.
11 Resuspend the pellet in 50 μ l of deionized H 2 O.
12 Measure absorbance of DNA at 260 nm and calculate concentration.

PROTOCOL 10.14 Electroporation of ES Cells with Targeting Vector

Equipment and reagents
• Incubator at 37 ◦ Cwith5%CO 2
• Water bath at 37 ◦ C