(a) Laboratory-grade water (800 μL).
(b) Stock 1.0 M NaOH (200 μL).
2. Invert the tube several times to mix. Use the fresh dilution
within 12 h.
3. Remove HT1 from À25
C to À15
C storage and thaw at
room temperature. Store at 2
C to 8
C until you are ready to
dilute denatured libraries.
4. Remove the tube of RSB (see Note 18) from À25
C to À15
C
storage and thaw at room temperature. Store thawed RSB at
2
C to 8
C until you are ready to dilute libraries.
5. Depending on the starting concentration of your library (see
Subheading 3.5), combine the following volume of freshly
diluted 0.2 M NaOH with the Library in a microcentrifuge
tube.
Starting library
concentration
Library
volume (μL)
0.2 M NaOH
volume (μL)
4 nM
5
5
2 nM
10
10
1 nM
20
20
0.5 nM
40
40
6. Vortex briefly and then centrifuge at 280 Â g for 1 min.
7. Incubate at room temperature for 5 min.
8. Add the following volume of 200 mM Tris–HCl, pH 7 to the
microfuge tube containing the Library:
(a) 4 nM Library: 5 μL Tris–HCl.
(b) 2 nM Library: 10 μL Tris–HCl.
(c) 1 nM Library: 20 μL Tris–HCl.
(d) 0.5 nM Library: 40 μL Tris–HCl.
9. Vortex briefly and then centrifuge at 280 Â g for 1 min.
10. Add the following volume of prechilled HT1 to the tube of
denatured library:
(a) 4 nM Library: 985 μL prechilled HT1.
(b) 2 nM Library: 970 μL prechilled HT1.
(c) 1 nM Library: 940 μL prechilled HT1.
(d) 0.5 nM Library: 880 μL prechilled HT1.
The result is a 20 pM denatured library.
11. Vortex briefly and then centrifuge at 280 Â g for 1 min.
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Shuyi Ma et al.