29:1, and 40 μL 20% SDS. Just before use, add 80 μL 10% APS
and 3.2 μL TEMED under a fume hood (see Note 10).
6. Stacking gel: 375 mM Tris–HCl pH 6.8, 5% Acrylamide:BisAcrylamide 29:1 (see Note 9), 0.1% SDS, 0.1% APS, 0.1%
TEMED (see Note 10). For one gel of 10 cm  7.5 cm  1 mm:
mix 3.3 mL H 2 O, 167 μL 3 M Tris pH 6.8, 0.5 mL 40%
Acrylamide:Bis-Acrylamide 29:1 and 20 μL 20% SDS. Just
before use, add 40 μL 10%APS and 4 μL TEMED (see Note
10).
2.2.3 Migration
1. 10Â Migration buffer: 0.25 M Tris base, 1.92 M Glycine, 1%
SDS. For 1 L: in a beaker, dissolve 144 g Glycine, 30 g of Tris
base and 50 mL of 20% SDS in H 2 O. Complete with H 2 O up
to 1 L with a graduated cylinder.
2. 1Â Migration buffer: 0.025 M Tris base, 0.192 M Glycine,
0.1% SDS. For 2 L: in a graduated cylinder, measure 200 mL of
10Â Migration buffer and complete with H 2 O up to 2 L.
2.2.4 Transfer
1. 10Â Transfer buffer: 1.92 M Glycine, 0.25 M glycine. For 1 L:
in a beaker, dissolve 144 g Glycine and 30 g of Tris base in
H 2 O. Complete with H 2 O up to 1 L with a graduated cylinder.
2. 1Â Transfer buffer: 10Â transfer buffer, 20% technical EtOH
(see Note 11). For 1 L: mix 100 mL of 10Â Transfer buffer
with 200 mL technical EtOH and complete with H 2 O up to
1 L.
3. Nitrocellulose membrane.
4. Whatman™ Grade 3MM CHR chromatography paper.
5. Red Ponceau S: 0.1% (w/v) Ponceau S in 5% (v/v) acetic acid.
2.2.5 Immunoblotting
1. 10Â PBS.
2. PBST: For 1 L, in a beaker, combine 100 mL of 10Â PBS into
approximately 800 mL of H 2 O. Add 2 mL of Tween20 with a
serological pipette (see Note 12) and complete with H 2 O up to
1 L in a graduated cylinder.
3. PBSTM (5% (w/v) skim milk in PBST): For 40 mL, dissolve
2 g of skim milk in 40 mL of PBST and mix thoroughly (see
Note 13). Make fresh (can be stored at 4
C for up to 1 day).
2.3 Material
for RT-qPCR
2.3.1 Reverse
Transcription
1. Mix 1: per tube, mix 1 μL of 10 mM dNTP (i.e., 0.5 mM final)
with 0.25 μL of 100 μM Random hexamer primers (i.e.,
1.25 μM final). Prepare freshly for N + 2 tubes and keep on ice.
2. Mix 2: per tube, mix 4 μL of 5Â RT buffer with 1 μL Maxima
H Minus enzyme (reverse transcriptase). Prepare freshly for
N + 2 tubes and keep on ice.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
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and 3.2 μL TEMED under a fume hood (see Note 10).
6. Stacking gel: 375 mM Tris–HCl pH 6.8, 5% Acrylamide:BisAcrylamide 29:1 (see Note 9), 0.1% SDS, 0.1% APS, 0.1%
TEMED (see Note 10). For one gel of 10 cm  7.5 cm  1 mm:
mix 3.3 mL H 2 O, 167 μL 3 M Tris pH 6.8, 0.5 mL 40%
Acrylamide:Bis-Acrylamide 29:1 and 20 μL 20% SDS. Just
before use, add 40 μL 10%APS and 4 μL TEMED (see Note
10).
2.2.3 Migration
1. 10Â Migration buffer: 0.25 M Tris base, 1.92 M Glycine, 1%
SDS. For 1 L: in a beaker, dissolve 144 g Glycine, 30 g of Tris
base and 50 mL of 20% SDS in H 2 O. Complete with H 2 O up
to 1 L with a graduated cylinder.
2. 1Â Migration buffer: 0.025 M Tris base, 0.192 M Glycine,
0.1% SDS. For 2 L: in a graduated cylinder, measure 200 mL of
10Â Migration buffer and complete with H 2 O up to 2 L.
2.2.4 Transfer
1. 10Â Transfer buffer: 1.92 M Glycine, 0.25 M glycine. For 1 L:
in a beaker, dissolve 144 g Glycine and 30 g of Tris base in
H 2 O. Complete with H 2 O up to 1 L with a graduated cylinder.
2. 1Â Transfer buffer: 10Â transfer buffer, 20% technical EtOH
(see Note 11). For 1 L: mix 100 mL of 10Â Transfer buffer
with 200 mL technical EtOH and complete with H 2 O up to
1 L.
3. Nitrocellulose membrane.
4. Whatman™ Grade 3MM CHR chromatography paper.
5. Red Ponceau S: 0.1% (w/v) Ponceau S in 5% (v/v) acetic acid.
2.2.5 Immunoblotting
1. 10Â PBS.
2. PBST: For 1 L, in a beaker, combine 100 mL of 10Â PBS into
approximately 800 mL of H 2 O. Add 2 mL of Tween20 with a
serological pipette (see Note 12) and complete with H 2 O up to
1 L in a graduated cylinder.
3. PBSTM (5% (w/v) skim milk in PBST): For 40 mL, dissolve
2 g of skim milk in 40 mL of PBST and mix thoroughly (see
Note 13). Make fresh (can be stored at 4
C for up to 1 day).
2.3 Material
for RT-qPCR
2.3.1 Reverse
Transcription
1. Mix 1: per tube, mix 1 μL of 10 mM dNTP (i.e., 0.5 mM final)
with 0.25 μL of 100 μM Random hexamer primers (i.e.,
1.25 μM final). Prepare freshly for N + 2 tubes and keep on ice.
2. Mix 2: per tube, mix 4 μL of 5Â RT buffer with 1 μL Maxima
H Minus enzyme (reverse transcriptase). Prepare freshly for
N + 2 tubes and keep on ice.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
357