Map of M13PK

Sequences are available upon order and request.

Preparation of M13PK double-stranded DNA

1. Culture TG1 or SS320 in 200ml of 2xYT medium containing 0.1% glucose at 37℃ and 250rpm OD600 = 0.8 to 1.0.
2. Infect the cells with phage M13PK at multiplicity of infection of 20:1 (phage-to-cell-ratio) for 15 min at 37℃.
3. Add 200ul of 15 mg/ml chloramphenicol in ethanol (final concentration, 15 μg/ml) to the culture. Incubate the culture an additional 2 hr.
4. Centrifuge 10 min at 6000g to harvest the cells. Prepare double-stranded DNA using TaKaRa Maxiprep kit (# 740414.50)

Amplification of M13PK phage

1. XL10-Gold was cultured overnight at 30°C in 2xYT containing 10 ug/ml tetracycline. 
2. 10ml 2xYT (containing 10ug/ml tetracycline) + 200ul overnight culture. Shaking at 250rpm and 30°C until OD600 =0.3 (about 3hours). 
3. Add M13PK phage at a multiplicity of infection of 20:1 (phage-to-cell-ratio).
4. Shake for 15min at 60rpm and 30°C, and then for 45min at 250rpm and 30°C.
5. Add the 10ml of infected culture to 1000 ml of prewarmed 2xYT containing 50ug/ml kanamycin, shake at 250rpm and 30°C until bacterial growth to the stationary phase (~24hours).
6. Purify M13PK phage from the supernatant by PEG/NaCl precipitation. 

Cloning strategy

1. KpnI and EagI digestion of M13PK DNA, and gel purification.
2. PCR amplification of insert with forward primer 5’-TTT AGT GGT ACC TTT CTA TTC TCA CTC G NNN NNN NNN NNN-3’ and reverse primer 5’- AAC AGT TTC GGC CGA NNN NNN NNN NNN-3’. 
3. KpnI and EagI digestion of the insert, and gel purification.
4. Ligation of KpnI and EagI digested M13PK and insert. 
5. DNA purification by ethanol precipitation.
6. Transformation of TG1 competent cells.

Production of peptide phage library

1. Add SOC to the transformed cells and incubate for 60 minutes at 30°C and 250rpm.
2. Add 2xYT containing 50ug/ml kanamycin to the culture and incubate overnight at 30°C and 250rpm.

Phage purification

1. 10000xg, 10min at 4C degree, collect the supernatant and trash the pellet.
2. Add 40g of PEG-8000 (4% w/v) and 30g of NaCl (3% w/v). After the solid is completely dissolved, keep in ice for 60min or overnight at 4°C.
3. Spin at 15000g for 15min at 4C. Discard the supernatant; spin down again and remove the remaining liquid. 
4. Resuspend the phage pellet in 25ml of TBS containing 0.5% BSA and 0.04% of NaN3. 
5. 20000g for 10min at 4°C to clear the supernatant.
6. Add 25ml of glycerol and mix completely. Store at -20°C for 2 years and at -80°C for long term.

Titration

1. XL10-Gold was cultured overnight at 30°C in 2xYT containing 10 ug/ml tetracycline.
2. 5ml 2xYT (containing 10ug/ml tetracycline) + 200ul overnight culture. Shaking at 250rpm and 30°C until OD600 around 0.5 (about 3hours).
3. Infect the E coli with serially diluted phage and make sure the cell number is greater than phage number.
4. Shake for 15min at 60rpm and 30°C, and then for 45min at 250rpm and 30°C.
5. Spread the culture on carbenicillin and kanamycin plates respectively, and put the plates at 37°C overnight. 
6. Count the colony number on the carbenicillin and kanamycin plates, and calculate the cfu/ml of the phagemid particles and phage particles respectively. 

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2. Buyers are NOT allowed to transfer or resell this product in any form.