Purify the nick-translated DNA by following steps 9–18.
9. Inactivate the DNase I by heating at 75
C for 10 min.
10. Clean-up the amine-modified DNA using a PCR purification
kit and elute in 100 μL of water (see Note 51).
11. Ethanol-precipitate the 100 μL nick-translated DNA by adding
10 μL of 3 M sodium acetate pH 5.2 and 275 μL of cold
absolute ethanol.
12. Leave at À20
C for 1 h minimum (overnight is better).
13. Centrifuge at 20,000 Â g for 30 min at 4
C.
14. Discard the supernatant, wash the pellet with 500 μL of 70%
cold absolute ethanol and centrifuge 15 min at 20,000 Â g at
4
C.
15. Discard the supernatant and let the residual liquid evaporate.
The pellet must be dry.
16. As it might stick to the tube wall after desiccation, resuspend
the nick-translated DNA pellet in 6 μL of water by vortexing
for 30 min at 37
C.
Table 3
References and characteristics of some Fosmids and BACs corresponding to EMT-related genes
References
Covered genes Protein
Size (bp) Coordinates (Dec. 2013 (GRCh38/hg38))
WI2-3715L22 CDH1
E-cadherin
45,918 chr16:68,747,043–68,792,960
WI2-1996A10 CDH1
E-cadherin
39,245 chr16:68,792,596–68,831,840
WI2-3409L5
CDH1
E-cadherin
39,413 chr16:68,721,690–68,761,102
WI2-3706F6
CDH1
E-cadherin
38,305 chr16:68,761,988–68,800,292
WI2-2774G15 CDH1
E-cadherin
34,708 chr16:68,796,994–68,831,701
WI2-3194M5
CDH2
N-cadherin
40,532 chr18:27,949,698–27,990,229
WI2-2158H2
CDH2
N-cadherin
36,745 chr18:27,990,131–28,026,875
WI2-3330A4
CDH2
N-cadherin
39,494 chr18:28,028,027–28,067,520
WI2-3413H15 CDH2
N-cadherin
43,383 chr18:28,073,240–28,116,622
WI2-2228E16 CDH2
N-cadherin
44,108 chr18:28,121,814–28,165,921
RP11-643F8
CDH2
N-cadherin 192,389 chr18:27,960,365–28,152,753
WI2-3327C11 OCLN
Occludin
40,741 chr5:69,505,597–69,546,337
WI2-2095C18 OCLN
Occludin
38,977 chr5:69,464,190–69,503,166
RP11-418N14 PGR
PGR
220,426 chr11:101,004,769–101,225,194
WI2-3907J23
VIM
Vimentin
38,741 chr10:17,220,428–17,259,168
PGR is our control gene
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
371
9. Inactivate the DNase I by heating at 75
C for 10 min.
10. Clean-up the amine-modified DNA using a PCR purification
kit and elute in 100 μL of water (see Note 51).
11. Ethanol-precipitate the 100 μL nick-translated DNA by adding
10 μL of 3 M sodium acetate pH 5.2 and 275 μL of cold
absolute ethanol.
12. Leave at À20
C for 1 h minimum (overnight is better).
13. Centrifuge at 20,000 Â g for 30 min at 4
C.
14. Discard the supernatant, wash the pellet with 500 μL of 70%
cold absolute ethanol and centrifuge 15 min at 20,000 Â g at
4
C.
15. Discard the supernatant and let the residual liquid evaporate.
The pellet must be dry.
16. As it might stick to the tube wall after desiccation, resuspend
the nick-translated DNA pellet in 6 μL of water by vortexing
for 30 min at 37
C.
Table 3
References and characteristics of some Fosmids and BACs corresponding to EMT-related genes
References
Covered genes Protein
Size (bp) Coordinates (Dec. 2013 (GRCh38/hg38))
WI2-3715L22 CDH1
E-cadherin
45,918 chr16:68,747,043–68,792,960
WI2-1996A10 CDH1
E-cadherin
39,245 chr16:68,792,596–68,831,840
WI2-3409L5
CDH1
E-cadherin
39,413 chr16:68,721,690–68,761,102
WI2-3706F6
CDH1
E-cadherin
38,305 chr16:68,761,988–68,800,292
WI2-2774G15 CDH1
E-cadherin
34,708 chr16:68,796,994–68,831,701
WI2-3194M5
CDH2
N-cadherin
40,532 chr18:27,949,698–27,990,229
WI2-2158H2
CDH2
N-cadherin
36,745 chr18:27,990,131–28,026,875
WI2-3330A4
CDH2
N-cadherin
39,494 chr18:28,028,027–28,067,520
WI2-3413H15 CDH2
N-cadherin
43,383 chr18:28,073,240–28,116,622
WI2-2228E16 CDH2
N-cadherin
44,108 chr18:28,121,814–28,165,921
RP11-643F8
CDH2
N-cadherin 192,389 chr18:27,960,365–28,152,753
WI2-3327C11 OCLN
Occludin
40,741 chr5:69,505,597–69,546,337
WI2-2095C18 OCLN
Occludin
38,977 chr5:69,464,190–69,503,166
RP11-418N14 PGR
PGR
220,426 chr11:101,004,769–101,225,194
WI2-3907J23
VIM
Vimentin
38,741 chr10:17,220,428–17,259,168
PGR is our control gene
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
371