7. 25 mg/ml kanamycin dissolved in deionized water. Sterile
filter.
8. 2YT/carb/kan/uridine: 2YT, 100 μg/ml carbenicillin, 25 μg/
ml kanamycin, 0.25 μg/ml uridine
9. PEG/NaCl: 20% PEG-8000 (w/v), 2.5 M NaCl (w/v). Sterile
filter.
10. PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM
KH 2 PO 4 . Adjust pH to 7.4 and autoclave.
11. QIAprep Spin M13 Kit.
12. MLB buffer: 1 M sodium perchlorate, 30% isopropanol (v/v).
13. Elution Buffer EB: 10 mM Tris–HCl, pH 8.5. Sterile filter.
14. 10Â TBE Buffer: 1 M Tris base, 1 M boric acid,
0.02 M EDTA.
2.3 In Vitro Synthesis
of Phagemid dsDNA
Library
1. Exonuclease I (ExoI).
2. 10Â TM buffer: 0.1 M MgCl 2 , 0.5 M Tris, pH 7.5.
3. 10 mM ATP, dissolve in H 2 O and sterile filter.
4. 100 mM dithiothreitol (DTT), dissolve in H 2 O and sterile
filter.
5. T4 polynucleotide kinase.
6. 10 mM dNTP mix
7. T7 DNA polymerase.
8. T4 DNA ligase.
9. FastDigest SmaI.
10. Qiagen QIA quick DNA purification kit.
11. QG buffer: 5.5 M guanidine thiocyanate, 20 mM Tris–HCl,
pH 6.6.
12. PE buffer: 10 mM Tris–HCl, 80% ethanol (v/v), pH 7.5.
13. Ultrapure water.
14. Agarose.
15. GelRed.
16. 10Â TBE Buffer.
2.4 Electroporation
and Amplification
of Library
1. E. coli SS320 preinfected with M13K07 helper phage prepared
as described elsewhere [11].
2. Gene Pulser electroporation device.
3. 0.1 cm gap electrode
4. SOC media: 0.5% yeast extract (w/v), 2% tryptone (w/v), 0.5%
NaCl (w/v), 0.2% KCl (w/v). Adjust pH to 7.0 with NaOH,
after autoclavation. Add 5.0 ml of autoclaved 2.0 M MgCl 2 and
20 ml of filter-sterilized 1.0 M glucose.
44
Susanne Lu ¨ chow et al.
filter.
8. 2YT/carb/kan/uridine: 2YT, 100 μg/ml carbenicillin, 25 μg/
ml kanamycin, 0.25 μg/ml uridine
9. PEG/NaCl: 20% PEG-8000 (w/v), 2.5 M NaCl (w/v). Sterile
filter.
10. PBS: 137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM
KH 2 PO 4 . Adjust pH to 7.4 and autoclave.
11. QIAprep Spin M13 Kit.
12. MLB buffer: 1 M sodium perchlorate, 30% isopropanol (v/v).
13. Elution Buffer EB: 10 mM Tris–HCl, pH 8.5. Sterile filter.
14. 10Â TBE Buffer: 1 M Tris base, 1 M boric acid,
0.02 M EDTA.
2.3 In Vitro Synthesis
of Phagemid dsDNA
Library
1. Exonuclease I (ExoI).
2. 10Â TM buffer: 0.1 M MgCl 2 , 0.5 M Tris, pH 7.5.
3. 10 mM ATP, dissolve in H 2 O and sterile filter.
4. 100 mM dithiothreitol (DTT), dissolve in H 2 O and sterile
filter.
5. T4 polynucleotide kinase.
6. 10 mM dNTP mix
7. T7 DNA polymerase.
8. T4 DNA ligase.
9. FastDigest SmaI.
10. Qiagen QIA quick DNA purification kit.
11. QG buffer: 5.5 M guanidine thiocyanate, 20 mM Tris–HCl,
pH 6.6.
12. PE buffer: 10 mM Tris–HCl, 80% ethanol (v/v), pH 7.5.
13. Ultrapure water.
14. Agarose.
15. GelRed.
16. 10Â TBE Buffer.
2.4 Electroporation
and Amplification
of Library
1. E. coli SS320 preinfected with M13K07 helper phage prepared
as described elsewhere [11].
2. Gene Pulser electroporation device.
3. 0.1 cm gap electrode
4. SOC media: 0.5% yeast extract (w/v), 2% tryptone (w/v), 0.5%
NaCl (w/v), 0.2% KCl (w/v). Adjust pH to 7.0 with NaOH,
after autoclavation. Add 5.0 ml of autoclaved 2.0 M MgCl 2 and
20 ml of filter-sterilized 1.0 M glucose.
44
Susanne Lu ¨ chow et al.