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Library Re-amplification

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For library in the form of phage particles:

  1. Inoculate 200 mL SB medium with 200 mL of TG1. Shake the culture at 300 rpm for 2 hr until the OD600=1.0.
  2. Add an aliquot of phage preparation (100 mL) to the culture and incubate at RT for 15 min. Add 40 mL of 100 mg/mL Ampicillin, shake the culture for 30 min at 37.
  3. Add 2 ml of M13KO7 helper phage (1012 to 1013 pfu/mL) and shake the culture for 2 hr at 37℃.
  4. Add 200 mL of 50 mg/mL Kanamycin and continue shaking 5 hr at 300 rpm and 37 .
  5. Spin at 3,000 g for 15 min at 4, transfer the supernatant to a clean centrifuge bottle and add 50 mL of PEG/NaCl, and incubate on ice for at least 1hr.
  6. Spin at 15,000 g for 15 min at 4 , discard the supernatant, and drain the bottle by inverting on a paper towel for at least 10 min, and wipe off the remaining liquid from the upper part of centrifuge bottle with a paper towel.
  7. Re-suspend the phage pellet in 2 mL of 1% (w/v) BSA in TBS by pipetting up and down along the side of centrifuge bottle. Transfer the suspension to a 2 mL micro-centrifuge tube. Spin at full speed in a micro-centrifuge for 5 min at 4 , and pass the supernatant through a 0.2 mm filter into a 2 mL micro-centrifuge tube. Store phage preparations at -20 .

For library in the form of phagemid DNA:

  1. Precipitate phagemid DNA by adding 1 mL of glycogen, 1 mL yeast tRNA (25 mg/mL), 3.5 mL of 3 M acetate, pH 5.2, and 80 mL of ethanol. Mix, and store at least 1hr at -80 .
  2. Spin at full speed in a micro-centrifuge for 15 min at 4oC. Remove and discard the supernatant; rinse the pellet twice with 1mL of 70% ethanol, drain inverted on a paper towel, and dry briefly. Dissolve the pellet in 15 mL of water.
  3. Place 1 cuvette on ice for 10 min. At the same time, thaw on ice, 300 mL of electro-competent E.coli.
  4. Use a 1-mL pipet tip with a snipped-off end to mix the electro-competent E.coli and the library by pipetting up and down once, and transfer to a cuvette. Store on ice for 1 min. Electro-transporate at 2.5kV, 25 mF, and 200Ω. Flush the cuvette immediately with 1mL and then twice with 2 mL of SOC medium at RT, and combine the 5 mL in a 50-mL flask. Shake at 250 rpm for 1hr at 37 .
  5. Add 10 mL of pre-warmed SB medium and 3 mL of 100 mg/mL Ampicillin. Shake the 15 mL culture at 250 rpm for 1hr at 37 , add 4.5 mL of 100 mg/mL Ampicillin, and shake for an additional hr at 250 rpm and 37 .
  6. Add 2 mL of M13KO7 helper phage (1012 to 1013 pfu/mL) and transfer to a 500 mL flask. Add 183 mL of pre-warmed SB medium and 92.5 mL of 100 mg/mL Ampicillin. Shake the 200 mL culture at 300 rpm for 1.5 hr at 37 .
  7. Add 200 mL of 50 mg/mL Kanamycin and continue shaking 5 hr at 300 rpm and 37 .
  8. Spin at 3,000 g for 15 min at 4 . Transfer the supernatant to a clean 500 mL flask and add 125 mL PEG/NaCl. Store on ice for 1hr.
  9. Spin at 15,000 g for 15 min at 4 . Discard the supernatant, drain the bottle by inverting on a paper towel for at least 10 min, and wipe off the remaining liquid from the upper part of the centrifuge bottle with a paper towel.
  10. Re-suspend the phage pellet in 2 mL of 1% (w/v) BSA in TBS by pipetting up and down along the side of centrifuge bottle. Transfer the suspension to a 2 mL micro-centrifuge tube. Spin at full speed in a micro-centrifuge for 5 min at 4 , and pass the supernatant through a 0.2 mm filter into a 2 mL micro-centrifuge tube. Store phage preparations at -20 .

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