49. If two values out of three are close, we consider the latest as
aberrant, most probably due to pipetting error.
50. For each fosmid or BAC associated nick translation reaction,
the ratio between DNase I nicking and DNA polymerase I
translation must be determined. Excess of DNase I will result
of short fragment sizes with low reaction yields (due to the
excessive DNA digestion) while lack of DNase I will result in a
poor aminoallyl-dUTP incorporation rate and large fragment
sizes (due to insufficient nicks quantity for the polymerase to
initiate translation). Users might want to titrate DNase I and
adjust the time to optimize nick translation reaction efficiency.
51. This step removes the trace of amines, which could interfere
with the subsequent labeling reaction. To increase the yield of
PCR purification, wash with 75% room temperature ethanol
(for 100 mL, mix 75 mL of absolute ethanol with 25 mL of
water), and elute two times in 50 μL water: put 50 μL of water
on the column, let stand for 2 min, centrifuge at the recommended speed for 5 min and repeat once.
52. The final concentration of the product can vary from 600 ng to
3 μg. If the quantity of DNA is below 1 μg, it is possible to pool
the product of two reactions and to evaporate the water to
reach a final volume of 5 μL.
53. Elution with 60 μL generally gives concentration of 10–15 ng/μL.
54. Correction factor ¼ (A 260 for the free dye)/(A max for the
free dye).
55. The labeling efficiency corresponds to the base to fluorescent
dye molecule ratio. Probes containing 16 to 30 bases per
fluorescent dye molecule will give a bright FISH signal whereas
lower degree of fluorophore incorporation might give undetectable FISH signals.
56. Here volumes are detailed for the use of 6-well plates with
22 Â 22 mm coverslips, you may adjust them if you use other
materials.
57. Carefully respect the 10 min incubation time as longer incubation may induce nucleus structure alterations.
58. The RNase A treatment will lead to the RNA digestion, which
will avoid unspecific hybridization of the probe.
59. HCl treatment will lead to the deproteinization of the DNA, a
key step of the DNA-FISH procedure. Times and concentration of the deproteinizing agent are critical and must be
observed rigorously. As they can vary depending on cell type
users may wish to empirically determine optimal conditions.
60. If cells are not immediately needed, skip the 1 h incubation at
room temperature and store the cells in 50% formamide at 4
C
for 1 month maximum. Surround the 6-well plate with parafilm before storing at 4
C.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
381
aberrant, most probably due to pipetting error.
50. For each fosmid or BAC associated nick translation reaction,
the ratio between DNase I nicking and DNA polymerase I
translation must be determined. Excess of DNase I will result
of short fragment sizes with low reaction yields (due to the
excessive DNA digestion) while lack of DNase I will result in a
poor aminoallyl-dUTP incorporation rate and large fragment
sizes (due to insufficient nicks quantity for the polymerase to
initiate translation). Users might want to titrate DNase I and
adjust the time to optimize nick translation reaction efficiency.
51. This step removes the trace of amines, which could interfere
with the subsequent labeling reaction. To increase the yield of
PCR purification, wash with 75% room temperature ethanol
(for 100 mL, mix 75 mL of absolute ethanol with 25 mL of
water), and elute two times in 50 μL water: put 50 μL of water
on the column, let stand for 2 min, centrifuge at the recommended speed for 5 min and repeat once.
52. The final concentration of the product can vary from 600 ng to
3 μg. If the quantity of DNA is below 1 μg, it is possible to pool
the product of two reactions and to evaporate the water to
reach a final volume of 5 μL.
53. Elution with 60 μL generally gives concentration of 10–15 ng/μL.
54. Correction factor ¼ (A 260 for the free dye)/(A max for the
free dye).
55. The labeling efficiency corresponds to the base to fluorescent
dye molecule ratio. Probes containing 16 to 30 bases per
fluorescent dye molecule will give a bright FISH signal whereas
lower degree of fluorophore incorporation might give undetectable FISH signals.
56. Here volumes are detailed for the use of 6-well plates with
22 Â 22 mm coverslips, you may adjust them if you use other
materials.
57. Carefully respect the 10 min incubation time as longer incubation may induce nucleus structure alterations.
58. The RNase A treatment will lead to the RNA digestion, which
will avoid unspecific hybridization of the probe.
59. HCl treatment will lead to the deproteinization of the DNA, a
key step of the DNA-FISH procedure. Times and concentration of the deproteinizing agent are critical and must be
observed rigorously. As they can vary depending on cell type
users may wish to empirically determine optimal conditions.
60. If cells are not immediately needed, skip the 1 h incubation at
room temperature and store the cells in 50% formamide at 4
C
for 1 month maximum. Surround the 6-well plate with parafilm before storing at 4
C.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
381