John M. S. Bartlett.pdf - Bio-Nica.info

Unknown Genomic Sequences 381
4. Centrifuge for 20 min at 4°C, remove the supernatant, wash the pellet with 500 µL of 80%
ethanol, and dry in the SpeedVac.
5. Dissolve DNA in 20 µL of water, transfer to a 500-µL PCR tube, and keep on ice.
6. Prepare a premix containing 5 µL of 10× Vent buffer, 1 µL of 10 mM dNTPs, 1 µL of
100 mM MgSO 4 (see Note 5), 1 µL of P2 solution, 1 µL of long-linker primer, and 19.5 µL
of water.
7. Immediately before use, add 1.5 µL of Vent DNA polymerase (3 U) to the premix and
mix by pipetting. Add premix to the PCR tube containing the DNA, then add two drops
of mineral oil, and keep on ice.
8. Transfer the tube to the thermal cycler (one droplet of mineral oil in sample holders)
preheated to 95°C, incubate for 2.5 min at 95°C, and subject to 18 cycles as follows: 95°C
for 1 min, 60°C for 2 min (see Note 5), and 76°C for 3 min. Allow an increase of 5 s/cycle
for the extension step, and end the cycling by a 10-min incubation at 76°C.
9. The PCR can be used immediately for the labeling step or stored frozen at –20°C.
3.7. Linker Tag Selection of the PCR Product
1. Resuspend the Dynabeads M-280 streptavidin well and take 50 µL (500 µg beads) into
a 1.5-mL reaction tube.
2. Concentrate in the magnetic rack (MPC, Dynal), and remove the supernatant (0.01%
BSA; see Note 6).
3. Wash the beads with 100 µL of PBS (pH 7.4), concentrate, and remove the supernatant.
Repeat this step.
4. Resuspend beads in 100 µL of PBS/0.01% BSA by pipetting up and down until a
homogenous suspension is achieved. Concentrate and remove supernatant.
5. Wash the beads with 100 µL of BW solution by pipetting up and down until no aggregates
are seen. Concentrate again. Repeat this step.
6. Finally resuspend the beads in 100 µL of BW solution. The beads are now ready to be
used.
7. Add the PCR to the beads and mix well. Avoid mineral oil, which spoils the magnetic
separation of beads (we do not recommend a chloroform extraction because traces of this
solvent adversely affects later steps in the reaction).
8. Incubate the tube on a rotating wheel at room temperature for 30 min. Assure that the beads
do not sediment in the tube but also avoid a spreading of the liquid over the entire tube
wall. A suitable agitation can be obtained by adjusting the rotation angle.
9. Concentrate beads and discard supernatant.
10. Wash the beads with 100 µL of BW solution.
3.8. Template Denaturation and Sequencing Reaction (see Note 7)
1. Resuspend beads well in 100 µL 150 mM NaOH by pipetting up and down. Incubate
at room temperature for 5 min with occasional gentle agitation, and then for 2 min
at 50°C.
2. Spin shortly to collect condensed liquid and beads trapped in the lid. Concentrate beads.
3. Remove the supernatant and resuspend the beads in 100 µL of 150 mM NaOH. Spin
shortly to collect all the NaOH solution and concentrate beads.
4. Discard supernatant, resuspend the beads in 100 µL of TE, pH 8.5, and concentrate again.
Repeat this wash with TE.
5. Resuspend the beads in 50 µL of Vent buffer and keep on ice.
6. Prepare four tubes labeled A, C, G, and T containing 3 µL of the respective termination
mixes (see Subheading 2.8.).