Post Panning Protocol 1: Rapid Purification of Single-Stranded DNA Templates for Sequencing Reactions
This rapid procedure produces template of sufficient purity for manual or automated dideoxy sequencing, without the use of phenol or chromatography. Alternatives would be to purify double-stranded DNA from infected cell pellets by standard mini prep or use PCR to make DNA sequencing templates.
- Amplify phage from individual plaques. Titer phage stock of interest to obtain plaques, see protocol Phage Titering. Use plates that are not older than 3 days, have less than 100 plaques and have been stored at 4°C, if not fresh. The next day, dilute an overnight culture of E. coli K12 ER2738 1:100 in LB. Dispense 1 ml of diluted culture into culture tubes, one for each clone to be sequenced. Use a sterile wooden stick or pipette tip to stab, i.e., pickup, a blue plaque and transfer phage particles to media in culture tube. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence. Shake cultures at 37°C at 250 rpm for 4.5–5 hours (no longer).
- Precipitate phage. Transfer cultures to microcentrifuge tubes, and microfuge at max speed (~20,000 x g) for 30 seconds. Transfer 500 μl of the phage-containing supernatant to a fresh microfuge tube. Add 200 μl of 20% PEG/2.5 M NaCl. Invert several times to mix and let stand for 10–20 minutes at room temperature.
Note: Supernatant from phage infected cultures may be stored at 4°C for several weeks or, with the addition of an equal volume of glycerol, at -20°C for several years. Phage with added PEG/NaCl may be stored for 1 week a 4°C.
- Precipitate nucleic acids. Microfuge at max speed (~20,000 x g) for 10 minutes and discard the supernatant. The phage pellet may not be visible. Suspend the pellet thoroughly in 100 μl of Iodide Buffer by vigorously tapping the tube. Add 250 μl of ethanol and incubate 10–20 minutes at room temperature. This short incubation at room temperature will preferentially precipitate single-stranded phage DNA, leaving most phage protein in solution. Spin in a microfuge at max speed for 10 minutes at 4°C and discard the supernatant. Wash the pellet with 0.5 ml of 70% ethanol (stored at -20°C), re-spin, discard the supernatant, and briefly dry the pellet under vacuum or for several hours on the bench top. Suspend the pellet in 30 μl of TE buffer; store at 4°C or -20°C.
- Quantify product by 1% TBE agarose gel electrophoresis. 5 μl of resuspended template should give a band of comparable intensity to 0.5 μg purified single-stranded M13mp18 DNA (NEB #N4040). Follow the commercial sequencing facility’s instructions to submit reactions for sequencing; typically, 0.5 μg DNA template and 1 pmol of primer are required.
- Follow the sequencing facility’s instructions to submit DNA for sequencing. Note, the raw sequence data generated with the -96gIII Sequencing Primer corresponds to the anticodon strand of the gIII region of the template. Obtain the reverse compliment of the data and compare the result against the top strand of the insert sequence shown in Figure 4 of the manual. Due to the design of the Ph.D. Phage Display Peptide Library oligonucleotides the third nucleotide of each codon in the randomized region should be either G or T. Note: TAG stop codons are suppressed by glutamine in ER2738 (glnV). If the library was amplified in this strain or any glnV (also known as supE) strain, TAG should be considered a glutamine codon when translating. Keep in mind that point mutations in a clone’s genome may present translations that are not exactly like the expected formats. Insertless and multiple insert clones may be observed. Sequences with stop codons in the middle are mis-translated or not accurately sequenced.