7. APS is subject to decomposition; therefore, it is best to prepare
it freshly. Though, preparing fresh each time can be tedious,
and you may therefore prepare 10 mL and store as 1 mL
aliquots at À20
C. In our hands, storing APS as such, and
avoiding many thaw/freeze cycles, showed no impact on gel
polymerization.
8. Keep TEMED tightly closed as it is subject to oxidation, which
leads to gradual loss of reactivity for polymerization. Store at
room temperature.
9. Keep the acrylamide solution protected from light.
10. 10% APS and TEMED are responsible for the gel polymerization. Due to its smell and toxicity, manipulating TEMED
under a fume hood is highly recommended.
11. In order to avoid evaporation, add EtOH in 1Â transfer
buffer only.
12. As Tween20 is very viscous, it is highly recommended to follow
this procedure in order to have the correct final concentration:
prepare a beaker with a magnetic stirrer, add the appropriate
volumes of 1Â PBS and H 2 O and start stirring, slowly withdraw Tween20 with a serological pipette up to 2 mL (do not
immerge the pipette too deep into the Tween20 as the Tween
will stick to the outside wall of the pipette, increasing the
volume of Tween you will be taking). Slowly release the
Tween20 into the solution. Leave the serological pipette into
the beaker with stirring and release Tween20 from time to time
into the solution until the pipette is empty. Then, pipet up and
down several times to rinse the pipette.
13. Taking skim milk is mandatory as the fat contained in other
milks can induce artifacts. Ensure that the milk is entirely
dissolved in the PBST before use, as undissolved milk can
provoke background spots on the membrane.
14. Prepare at least 30% more volume than needed in order to
account for the viscosity of the SYBR and the dead volume of
the electronic pipette used for dispensing.
15. Perform the final elution in water and not in TE buffer because
the presence of EDTA will inhibit the DNase I action during
nick translation reaction.
16. Paraformaldehyde is toxic and should be disposed using appropriate hazardous waste streams.
17. Triton X-100 is highly viscous and sticks to the pipet tip walls.
In order to reach a dilution as precise as possible, cut the
bottom end of a 1000 μL-pipette tip before taking 500 μL of
Triton X-100 very slowly. Distribute in 20 mL of 1Â PBS, wait
for the Triton to go down by gravity before releasing again,
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
377
it freshly. Though, preparing fresh each time can be tedious,
and you may therefore prepare 10 mL and store as 1 mL
aliquots at À20
C. In our hands, storing APS as such, and
avoiding many thaw/freeze cycles, showed no impact on gel
polymerization.
8. Keep TEMED tightly closed as it is subject to oxidation, which
leads to gradual loss of reactivity for polymerization. Store at
room temperature.
9. Keep the acrylamide solution protected from light.
10. 10% APS and TEMED are responsible for the gel polymerization. Due to its smell and toxicity, manipulating TEMED
under a fume hood is highly recommended.
11. In order to avoid evaporation, add EtOH in 1Â transfer
buffer only.
12. As Tween20 is very viscous, it is highly recommended to follow
this procedure in order to have the correct final concentration:
prepare a beaker with a magnetic stirrer, add the appropriate
volumes of 1Â PBS and H 2 O and start stirring, slowly withdraw Tween20 with a serological pipette up to 2 mL (do not
immerge the pipette too deep into the Tween20 as the Tween
will stick to the outside wall of the pipette, increasing the
volume of Tween you will be taking). Slowly release the
Tween20 into the solution. Leave the serological pipette into
the beaker with stirring and release Tween20 from time to time
into the solution until the pipette is empty. Then, pipet up and
down several times to rinse the pipette.
13. Taking skim milk is mandatory as the fat contained in other
milks can induce artifacts. Ensure that the milk is entirely
dissolved in the PBST before use, as undissolved milk can
provoke background spots on the membrane.
14. Prepare at least 30% more volume than needed in order to
account for the viscosity of the SYBR and the dead volume of
the electronic pipette used for dispensing.
15. Perform the final elution in water and not in TE buffer because
the presence of EDTA will inhibit the DNase I action during
nick translation reaction.
16. Paraformaldehyde is toxic and should be disposed using appropriate hazardous waste streams.
17. Triton X-100 is highly viscous and sticks to the pipet tip walls.
In order to reach a dilution as precise as possible, cut the
bottom end of a 1000 μL-pipette tip before taking 500 μL of
Triton X-100 very slowly. Distribute in 20 mL of 1Â PBS, wait
for the Triton to go down by gravity before releasing again,
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
377