9. Centrifuge at 12,000 g for 5 min at 4
C. Mix supernatant to
previous one of each sample. Add 1 mL ethanol. Mix well and
incubate it at À20
C overnight.
10. Collect the precipitation by centrifuging at 21,130 g for
15 min. Wash the pellet with 75% ethanol and centrifuge
again at 21,130 g for 5 min.
11. Discard the supernatant and air-dry the pellet for 5 min.
12. Dissolve the pellet in 100 μL RNase-free water. Measure the
concentration by a nano drop. Add water to a final concentration of 50 μM.
3.2 Primer
Purification
Commercial oligonucleotides contain accumulated impurities and
truncated forms during the synthesis process. The default desalting
purification can only remove excess salt but not truncated
sequences. While the commercial PAGE purification can be additional expense, here we show how to purify primers through a
home-developed PAGE method.
1. Assemble a gel cast. Prepare 10% urea-polyacrylamide gel
(Table 2) with the size of 16 cmÂ16 cmÂ1.5 mm (The gel
volume is about 40 mL/gel).
2. Add water to commercial purchased primers and adapters (see
Note 3) to a final concentration of 100 μM. Take 50 μL and
mix very well with 50 μL FA buffer.
3. Assemble cast with electrophoresis tank. Use clips to fix a piece
of aluminum pad with thin glass plate of the cast to share heat
when running electrophoresis. Fill in the top and bottom tank
with 1Â TBE buffer.
4. Use a needle and a syringe to completely clean urea from wells.
Use a curved needle and a syringe to remove bubbles at the
bottom.
5. Load samples into wells, and split one sample into two wells if
necessary. Leave one empty well between different samples. Fill
empty wells with FA buffer to make sure the samples run
straight.
6. Run electrophoresis at 500 V or 200 mA or 35 W per gel for
1 h.
7. Dismantle the gel cast and gently transfer the gel to plastic
wrap. Put the gel on the top of thick glass plate matching the
labels. Use a UV lamp (254 nm) to check the signal of DNA.
Draw the position along the outline of the bands. Then use a
clean blade to cut the gel along the sketch. Chop that region
into small pieces and carefully transfer them to a 1.5 mL tube
(see Note 4). Use clean water to rinse and kim wipe to clean the
blade between samples.
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Di Sun et al.
C. Mix supernatant to
previous one of each sample. Add 1 mL ethanol. Mix well and
incubate it at À20
C overnight.
10. Collect the precipitation by centrifuging at 21,130 g for
15 min. Wash the pellet with 75% ethanol and centrifuge
again at 21,130 g for 5 min.
11. Discard the supernatant and air-dry the pellet for 5 min.
12. Dissolve the pellet in 100 μL RNase-free water. Measure the
concentration by a nano drop. Add water to a final concentration of 50 μM.
3.2 Primer
Purification
Commercial oligonucleotides contain accumulated impurities and
truncated forms during the synthesis process. The default desalting
purification can only remove excess salt but not truncated
sequences. While the commercial PAGE purification can be additional expense, here we show how to purify primers through a
home-developed PAGE method.
1. Assemble a gel cast. Prepare 10% urea-polyacrylamide gel
(Table 2) with the size of 16 cmÂ16 cmÂ1.5 mm (The gel
volume is about 40 mL/gel).
2. Add water to commercial purchased primers and adapters (see
Note 3) to a final concentration of 100 μM. Take 50 μL and
mix very well with 50 μL FA buffer.
3. Assemble cast with electrophoresis tank. Use clips to fix a piece
of aluminum pad with thin glass plate of the cast to share heat
when running electrophoresis. Fill in the top and bottom tank
with 1Â TBE buffer.
4. Use a needle and a syringe to completely clean urea from wells.
Use a curved needle and a syringe to remove bubbles at the
bottom.
5. Load samples into wells, and split one sample into two wells if
necessary. Leave one empty well between different samples. Fill
empty wells with FA buffer to make sure the samples run
straight.
6. Run electrophoresis at 500 V or 200 mA or 35 W per gel for
1 h.
7. Dismantle the gel cast and gently transfer the gel to plastic
wrap. Put the gel on the top of thick glass plate matching the
labels. Use a UV lamp (254 nm) to check the signal of DNA.
Draw the position along the outline of the bands. Then use a
clean blade to cut the gel along the sketch. Chop that region
into small pieces and carefully transfer them to a 1.5 mL tube
(see Note 4). Use clean water to rinse and kim wipe to clean the
blade between samples.
238
Di Sun et al.