236
HEINZ-ULRICH G. WEIER
on a UV trans-illuminator, cut out a gel slice at the corresponding position
from the unstained half and proceed as described in z.
2. Recovery of high molecular weight DNA from LMP agarose gel slices
1. Melt the gel completely by incubating it for 10 min at 85°C.
2. Transfer the molten agarose to a 43°C water bath.
3. Add 1 J.ll ~-agarase for every 25 J.ll of molten agarose.
4. Incubate at 43°C for 2 h.
5. Add an equal volume of 200 mM NaCl.
6. Store the sample at 4°C until use.
3. Genomic DNA
Genomic high molecular weight (HMW) DNA is isolated from exponentially growing human cells such as the C32 melanoma cell line (ATCC) or
diploid fibroblast cells using standard procedures. Briefly, about 5x 10
5
cells are washed in PBS. The cells are then resuspended in 0.5 ml of
PBS and mixed with 1.2% low melting point agarose previously melted
in PBS and allowed to cool down to 43°C. Aliquots of 100J.ll are dispensed
into plug molds and allowed to set for 30 min at 4°C. Agarose plugs are
then placed into DB 0.5 solution and incubated overnight at 50°C. Next,
plugs are washed 4-6 times for 30 min each in 50 mM Tris HCl, ImM EDTA
and stored at 4°C. The HMW DNA is released by digestion of the plugs
with ~-agarase according to the manufacturer's instructions (NEB) as described above.
Subprotocol 3
Immobilization and stretching of DNA molecules
The correct immobilization of DNA molecules is important for the successful stretching as well as the minimization of DNA loss during denaturation and hybridization. We have used different methods for binding
and stretching of DNA on APS-pretreated surfaces. The quality of the resulting DNA fibers on glass or mica surfaces (Hu et aI., 1996) appears to be
HEINZ-ULRICH G. WEIER
on a UV trans-illuminator, cut out a gel slice at the corresponding position
from the unstained half and proceed as described in z.
2. Recovery of high molecular weight DNA from LMP agarose gel slices
1. Melt the gel completely by incubating it for 10 min at 85°C.
2. Transfer the molten agarose to a 43°C water bath.
3. Add 1 J.ll ~-agarase for every 25 J.ll of molten agarose.
4. Incubate at 43°C for 2 h.
5. Add an equal volume of 200 mM NaCl.
6. Store the sample at 4°C until use.
3. Genomic DNA
Genomic high molecular weight (HMW) DNA is isolated from exponentially growing human cells such as the C32 melanoma cell line (ATCC) or
diploid fibroblast cells using standard procedures. Briefly, about 5x 10
5
cells are washed in PBS. The cells are then resuspended in 0.5 ml of
PBS and mixed with 1.2% low melting point agarose previously melted
in PBS and allowed to cool down to 43°C. Aliquots of 100J.ll are dispensed
into plug molds and allowed to set for 30 min at 4°C. Agarose plugs are
then placed into DB 0.5 solution and incubated overnight at 50°C. Next,
plugs are washed 4-6 times for 30 min each in 50 mM Tris HCl, ImM EDTA
and stored at 4°C. The HMW DNA is released by digestion of the plugs
with ~-agarase according to the manufacturer's instructions (NEB) as described above.
Subprotocol 3
Immobilization and stretching of DNA molecules
The correct immobilization of DNA molecules is important for the successful stretching as well as the minimization of DNA loss during denaturation and hybridization. We have used different methods for binding
and stretching of DNA on APS-pretreated surfaces. The quality of the resulting DNA fibers on glass or mica surfaces (Hu et aI., 1996) appears to be