270
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
2. Construct a linear 10% to 40% sucrose density gradient according to the
manufacturer's guidelines (several gradient makers are commercially
available) using either a SW-28 or SW-41 centrifuge tube.
3. Heat the digested DNA at 65°C for 5 min to dissociate DNA aggregates.
Gently pour no more than 0.5 mg (SW-28) or 0.2 mg (SW-41) ofheated
genomic DNA solution onto the top of the sucrose density gradient.
Lambda vector DNA should not be used in amounts exceeding 50 f.lg
(SW-28) or 20 J.lg (SW-41).
4. Centrifuge the gradient tubes for 16- 18 hr for cosmid-sized inserts and
24 h for bacteriophage-sized inserts. Spinat 113,000 x g (SW -28 rotor) or
154,000 x g (SW-41 rotor) at 20°C.
5. After centrifugation is completed, 750 f.!l aliquots are collected from the
bottom of the tube using a capillary tube. The size range of each portion is
evaluated by gel electrophoresis.
6. The desired fractions are subdivided into 4 microcentrifuge tubes containing -180 f.!l/ each. 320 f.ll of water and 1 ml ethanol are added to the
samples, and the tubes are placed at - 20°C overnight to precipitate the
samples.
7. At this point, the samples can be stored indefinitely at - 80°C for future
use or redissolved in 100 f.ll ofTE buffer (10 mM Tris-Cl (pH 7.5), 1 mM
EDTA, (pH 8.0)) or autoclaved deionized water.
Additional information related to these and other protocols can be found in
Ausubel et al. 1991.
Comments
Library complexity
A library is described as being "deep" or "representative" when it contains
all fragments from a given DNA source. No library has yet been produced
which includes a complete set of overlapping clones. As discussed previously, some of the reasons for this are:
• repetitive sequences that are unstable in prokaryotic or yeast hosts or
that are prone to recombination (rearrangements)
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
2. Construct a linear 10% to 40% sucrose density gradient according to the
manufacturer's guidelines (several gradient makers are commercially
available) using either a SW-28 or SW-41 centrifuge tube.
3. Heat the digested DNA at 65°C for 5 min to dissociate DNA aggregates.
Gently pour no more than 0.5 mg (SW-28) or 0.2 mg (SW-41) ofheated
genomic DNA solution onto the top of the sucrose density gradient.
Lambda vector DNA should not be used in amounts exceeding 50 f.lg
(SW-28) or 20 J.lg (SW-41).
4. Centrifuge the gradient tubes for 16- 18 hr for cosmid-sized inserts and
24 h for bacteriophage-sized inserts. Spinat 113,000 x g (SW -28 rotor) or
154,000 x g (SW-41 rotor) at 20°C.
5. After centrifugation is completed, 750 f.!l aliquots are collected from the
bottom of the tube using a capillary tube. The size range of each portion is
evaluated by gel electrophoresis.
6. The desired fractions are subdivided into 4 microcentrifuge tubes containing -180 f.!l/ each. 320 f.ll of water and 1 ml ethanol are added to the
samples, and the tubes are placed at - 20°C overnight to precipitate the
samples.
7. At this point, the samples can be stored indefinitely at - 80°C for future
use or redissolved in 100 f.ll ofTE buffer (10 mM Tris-Cl (pH 7.5), 1 mM
EDTA, (pH 8.0)) or autoclaved deionized water.
Additional information related to these and other protocols can be found in
Ausubel et al. 1991.
Comments
Library complexity
A library is described as being "deep" or "representative" when it contains
all fragments from a given DNA source. No library has yet been produced
which includes a complete set of overlapping clones. As discussed previously, some of the reasons for this are:
• repetitive sequences that are unstable in prokaryotic or yeast hosts or
that are prone to recombination (rearrangements)