one at the size of the ssDNA which is DNA that has not been
converted, another at the size of the CCC-dsDNA product
correctly extended and ligated, and possibly a band with unligated dsDNA higher up on the gel.
17. The desalted CCC-dsDNA can directly be used for electroporation to create a phage library, or frozen for later use.
3.5 Electroporation
of Phagemid Library
and Amplification
of Phage Library
1. Chill 100 μl CCC-dsDNA and a 0.1 cm gap electrode on ice.
2. Thaw an aliquot of electrocompetent E. coli SS320 preinfected
with M13K07 on ice. Add 150 μl of the bacteria to the DNA
and mix carefully to avoid creating bubbles.
3. Transfer the mixture to the prechilled cuvette and electroporate the mixture with a Gene Pulser with the settings 1.8 kV
200 Ω and 25 μF. Take note of the time constant. A time
constant in the range of 3.5–4 is typically indicative of a successful transformation.
4. Immediately add 1 ml prewarmed SOC media to the cuvette
and transfer the culture to 10 ml SOC media in a 250 ml flask.
Rinse the cuvette with SOC media two times (pipet up and
down a couple of times to mix) and add it to the culture. Add
additional SOC medium to a final volume of 25 ml.
5. Incubate for 30 min at 37
C with shaking at 200 rpm.
6. To determine the efficiency of the electroporation, make three
10Â serial dilutions, for 12 dilution steps of the 25 ml culture,
exactly 30 min after electroporation. Spot 5 μl of each serial
dilution on an LB/carb plate and incubate at 37
C overnight.
Count the number of colonies and calculate backwards to
determine the number of colony-forming units in the 25 ml
SOC media. This sets the upper limit of the library size. When
making ProP-PD libraries, the aim is to have at least 100-1000
times more transformants than the number of individual
sequences in the library design to ensure a comprehensive
coverage.
7. Transfer the culture to a 2 l flask containing 500 ml 2YT/carbenicillin/kanamycin medium and incubate it for at least 16 h
at 37
C shaking and 200 rpm.
8. Centrifuge the culture at 16,000 Â g for 10 min in a Beckman
JA14 rotor or equivalent.
9. Transfer the supernatant containing the phage library to a new
centrifuge tube containing 1/5 of the final volume of PEG/NaCl solution for phage precipitation.
10. Incubate 5 min at room temperature and spin it at 16,000 Â g
for 10 min. Remove the supernatant.
50
Susanne Lu ¨ chow et al.
converted, another at the size of the CCC-dsDNA product
correctly extended and ligated, and possibly a band with unligated dsDNA higher up on the gel.
17. The desalted CCC-dsDNA can directly be used for electroporation to create a phage library, or frozen for later use.
3.5 Electroporation
of Phagemid Library
and Amplification
of Phage Library
1. Chill 100 μl CCC-dsDNA and a 0.1 cm gap electrode on ice.
2. Thaw an aliquot of electrocompetent E. coli SS320 preinfected
with M13K07 on ice. Add 150 μl of the bacteria to the DNA
and mix carefully to avoid creating bubbles.
3. Transfer the mixture to the prechilled cuvette and electroporate the mixture with a Gene Pulser with the settings 1.8 kV
200 Ω and 25 μF. Take note of the time constant. A time
constant in the range of 3.5–4 is typically indicative of a successful transformation.
4. Immediately add 1 ml prewarmed SOC media to the cuvette
and transfer the culture to 10 ml SOC media in a 250 ml flask.
Rinse the cuvette with SOC media two times (pipet up and
down a couple of times to mix) and add it to the culture. Add
additional SOC medium to a final volume of 25 ml.
5. Incubate for 30 min at 37
C with shaking at 200 rpm.
6. To determine the efficiency of the electroporation, make three
10Â serial dilutions, for 12 dilution steps of the 25 ml culture,
exactly 30 min after electroporation. Spot 5 μl of each serial
dilution on an LB/carb plate and incubate at 37
C overnight.
Count the number of colonies and calculate backwards to
determine the number of colony-forming units in the 25 ml
SOC media. This sets the upper limit of the library size. When
making ProP-PD libraries, the aim is to have at least 100-1000
times more transformants than the number of individual
sequences in the library design to ensure a comprehensive
coverage.
7. Transfer the culture to a 2 l flask containing 500 ml 2YT/carbenicillin/kanamycin medium and incubate it for at least 16 h
at 37
C shaking and 200 rpm.
8. Centrifuge the culture at 16,000 Â g for 10 min in a Beckman
JA14 rotor or equivalent.
9. Transfer the supernatant containing the phage library to a new
centrifuge tube containing 1/5 of the final volume of PEG/NaCl solution for phage precipitation.
10. Incubate 5 min at room temperature and spin it at 16,000 Â g
for 10 min. Remove the supernatant.
50
Susanne Lu ¨ chow et al.