and 1 μL glycogen in turn. Mix well and precipitate at À80
C
for at least 30 min, or À20
C overnight.
6. Centrifuge at maximum speed at 4
C for 15 min. Carefully
remove the supernatant by pipetting without touching the
pellet.
7. Wash the pellet with 500 μL 75% ethanol. Centrifuge at
13,800 g at 4
C for 5 min. Discard ethanol. Spin down quickly
and carefully remove the residual ethanol through pipetting.
8. Keep the tubes on a neat bench and air-dry for at most 5 min.
Then resuspend the pellet with 2.5 μL RNase-free water.
3.3.2 T4 PNK Treatment
1. Prepare PNK reaction system for each marker on ice as follows:
1 μL commercially purchased or home-made marker (Table 2)
solution dissolved in RNase-free water (10 μM), 1 μL ten-fold
NEB PNK buffer, 5 μL of γ-P
32
-ATP (3000 Ci/mM or
10 mCi/mL), 0.5 μL RNase inhibitor (final concentration
1 U/μL), 1 μl T4 polynucleotide kinase, RNase-free water
1.5 μL. Or add the following reagents to each of the resuspended markers from CIP treatment: 1 μL ten-fold NEB PNK
buffer, 5 μL of γ-ATP
32 (3000 Ci/mM or 10 mCi/mL),
0.5 μL RNase inhibitor (final concentration 1 U/μL), and
1 μL T4 polynucleotide kinase. Mix well and incubate at
37
C for 2 h.
2. Add 230 μL RNase-free water to each of the above reaction
mixture, respectively. Then recover markers by repeating the
steps 3–7 described in Subheading 3.3.1.
3. Resuspend the pellet with 20 μL FA buffer, respectively.
4. Assemble cast with electrophoresis tank. Use clips to attach a
piece of aluminum pad on the surface of the thin glass plate to
spread heat when running electrophoresis. Fill in the top and
bottom tank with 1X TBE buffer made with RNase-free water.
5. Denature samples at 95
C for 3 min, then place them on ice for
2 min, followed by a quick spin. Load samples to 15% ureapolyacrylamide gel (Table 2) with the size of 16 cm Â16 cmÂ
0.8 mm (the gel volume is about 25 mL/gel).
6. Use a needle and a syringe to clean wells to make sure no urea
remains. Use a curved needle and a syringe to remove bubbles
at the bottom.
7. Load samples and run electrophoresis at 500 V or 200 mA or
35 W per gel for 1 h.
8. Dismantle the gel cast and leave the gel on the top of thick glass
plate matching the labels. Implant three tiny radioactive gel
pieces asymmetrically at three of four corners of the gel so one
can align the gel to the phosphorimager paper printout at the
next step. Radioactive gel pieces can be collected from the gel
240
Di Sun et al.
C
for at least 30 min, or À20
C overnight.
6. Centrifuge at maximum speed at 4
C for 15 min. Carefully
remove the supernatant by pipetting without touching the
pellet.
7. Wash the pellet with 500 μL 75% ethanol. Centrifuge at
13,800 g at 4
C for 5 min. Discard ethanol. Spin down quickly
and carefully remove the residual ethanol through pipetting.
8. Keep the tubes on a neat bench and air-dry for at most 5 min.
Then resuspend the pellet with 2.5 μL RNase-free water.
3.3.2 T4 PNK Treatment
1. Prepare PNK reaction system for each marker on ice as follows:
1 μL commercially purchased or home-made marker (Table 2)
solution dissolved in RNase-free water (10 μM), 1 μL ten-fold
NEB PNK buffer, 5 μL of γ-P
32
-ATP (3000 Ci/mM or
10 mCi/mL), 0.5 μL RNase inhibitor (final concentration
1 U/μL), 1 μl T4 polynucleotide kinase, RNase-free water
1.5 μL. Or add the following reagents to each of the resuspended markers from CIP treatment: 1 μL ten-fold NEB PNK
buffer, 5 μL of γ-ATP
32 (3000 Ci/mM or 10 mCi/mL),
0.5 μL RNase inhibitor (final concentration 1 U/μL), and
1 μL T4 polynucleotide kinase. Mix well and incubate at
37
C for 2 h.
2. Add 230 μL RNase-free water to each of the above reaction
mixture, respectively. Then recover markers by repeating the
steps 3–7 described in Subheading 3.3.1.
3. Resuspend the pellet with 20 μL FA buffer, respectively.
4. Assemble cast with electrophoresis tank. Use clips to attach a
piece of aluminum pad on the surface of the thin glass plate to
spread heat when running electrophoresis. Fill in the top and
bottom tank with 1X TBE buffer made with RNase-free water.
5. Denature samples at 95
C for 3 min, then place them on ice for
2 min, followed by a quick spin. Load samples to 15% ureapolyacrylamide gel (Table 2) with the size of 16 cm Â16 cmÂ
0.8 mm (the gel volume is about 25 mL/gel).
6. Use a needle and a syringe to clean wells to make sure no urea
remains. Use a curved needle and a syringe to remove bubbles
at the bottom.
7. Load samples and run electrophoresis at 500 V or 200 mA or
35 W per gel for 1 h.
8. Dismantle the gel cast and leave the gel on the top of thick glass
plate matching the labels. Implant three tiny radioactive gel
pieces asymmetrically at three of four corners of the gel so one
can align the gel to the phosphorimager paper printout at the
next step. Radioactive gel pieces can be collected from the gel
240
Di Sun et al.