6. Centrifuge the sample for 10 min at 12,000 Â g to pellet the
precipitated phages. Decant the supernatant, and spin briefly
for 2 min at 2000 Â g to concentrate the phage pellet. Aspirate
the remaining supernatant.
7. Resuspend the phage pellet in 500 μl PBS and transfer it to a
microcentrifuge tube and centrifuge it at 14,000 Â g for 5 min
to remove insoluble debris and transfer the supernatant to a
new microcentrifuge tube.
8. Add 7 μl buffer MP, mix and incubate at room temperature for
2 min.
9. Add the sample to a spin column in a 2 ml microcentrifuge
tube, centrifuge at 6000 Â g for 30 s and discard the flowthrough. The phage remains bound to the spin column matrix.
10. Add 0.7 ml MLB buffer to the spin column and centrifuge at
6000 Â g for 30 s and discard the flow through.
11. Add 0.7 ml MLB buffer to the spin column, incubate for 1 min
and centrifuge at 6000 Â g for 30 s and discard the flow
through. The DNA is now bound to the column matrix.
12. Add 0.7 ml PE buffer to the spin column and centrifuge at
6000 Â g for 30 s and discard the flow through.
13. Repeat step 12.
14. Centrifuge the empty tube at 6000 Â g for 30 s to remove
residual buffer.
15. Transfer the column to a fresh 1.5 ml microcentrifuge tube and
add 100 μl buffer EB to the center of the membrane.
16. Incubate for 10 min at room temperature and then centrifuge
at 6000 Â g for 30 s. The eluent contains the purified
dU-ssDNA.
17. Analyze 1 μl of the DNA by 1% (w/v) agarose gel electrophoresis using 1xTBE buffer and 1:10,000 GelRed.
18. Determine the DNA concentration by absorbance at 260 nm
(A 260 ¼ 1.0 for 33 ng/ml of ssDNA) using a NanoDrop or
equivalent.
3.4 In Vitro Synthesis
of the Phagemid
dsDNA Library
In this process you will turn the amplified oligonucleotide pool and
the dU-ssDNA in to a heteroduplex covalently closed circular
double stranded DNA (CCC-dsDNA). This will be done in three
steps: 5
0 phosphorylation of the PCR amplified oligonucleotide
pool, annealing to the circular dU-ssDNA and enzymatic synthesis
of the second strand to create CCC-dsDNA. This is a scaled-up
version of a published method by Kunkel and coworkers [13], and
modified for the use of PCR amplified oligonucleotide library.
Before phosphorylating the PCR amplified oligonucleotide pool,
you will remove the remaining single stranded PCR primers by
treating the sample with ExoI.
48
Susanne Lu ¨ chow et al.
precipitated phages. Decant the supernatant, and spin briefly
for 2 min at 2000 Â g to concentrate the phage pellet. Aspirate
the remaining supernatant.
7. Resuspend the phage pellet in 500 μl PBS and transfer it to a
microcentrifuge tube and centrifuge it at 14,000 Â g for 5 min
to remove insoluble debris and transfer the supernatant to a
new microcentrifuge tube.
8. Add 7 μl buffer MP, mix and incubate at room temperature for
2 min.
9. Add the sample to a spin column in a 2 ml microcentrifuge
tube, centrifuge at 6000 Â g for 30 s and discard the flowthrough. The phage remains bound to the spin column matrix.
10. Add 0.7 ml MLB buffer to the spin column and centrifuge at
6000 Â g for 30 s and discard the flow through.
11. Add 0.7 ml MLB buffer to the spin column, incubate for 1 min
and centrifuge at 6000 Â g for 30 s and discard the flow
through. The DNA is now bound to the column matrix.
12. Add 0.7 ml PE buffer to the spin column and centrifuge at
6000 Â g for 30 s and discard the flow through.
13. Repeat step 12.
14. Centrifuge the empty tube at 6000 Â g for 30 s to remove
residual buffer.
15. Transfer the column to a fresh 1.5 ml microcentrifuge tube and
add 100 μl buffer EB to the center of the membrane.
16. Incubate for 10 min at room temperature and then centrifuge
at 6000 Â g for 30 s. The eluent contains the purified
dU-ssDNA.
17. Analyze 1 μl of the DNA by 1% (w/v) agarose gel electrophoresis using 1xTBE buffer and 1:10,000 GelRed.
18. Determine the DNA concentration by absorbance at 260 nm
(A 260 ¼ 1.0 for 33 ng/ml of ssDNA) using a NanoDrop or
equivalent.
3.4 In Vitro Synthesis
of the Phagemid
dsDNA Library
In this process you will turn the amplified oligonucleotide pool and
the dU-ssDNA in to a heteroduplex covalently closed circular
double stranded DNA (CCC-dsDNA). This will be done in three
steps: 5
0 phosphorylation of the PCR amplified oligonucleotide
pool, annealing to the circular dU-ssDNA and enzymatic synthesis
of the second strand to create CCC-dsDNA. This is a scaled-up
version of a published method by Kunkel and coworkers [13], and
modified for the use of PCR amplified oligonucleotide library.
Before phosphorylating the PCR amplified oligonucleotide pool,
you will remove the remaining single stranded PCR primers by
treating the sample with ExoI.
48
Susanne Lu ¨ chow et al.