Amplifying T7:
The following protocol is just for the Plate Lysate amplification. It is taken almost verbatim from the Novagen T7 system select manual (pg 13). For the liquid lysate amplification used to amplify a large number of phage (> 1E7), consult pg 14 of the manual.
A single round of amplification is necessary prior to biopanning. There are two basic methods for library amplification depending on the size and complexity of the library. For a cDNA or other library where the total number of primary recombinants is less than 5E6, the plate lysate is the preferred method. When amplifying larger libraries the liquid lysate method is required for logistical reasons.
1. Inoculate 50 ml TB with 1 ml overnight culture of the appropriate host. Shake at 37°C until the OD600 reaches 0.6--1.0. If using a 5615 host strain, add IPTG to 1 mM when the OD600 reaches 0.5 and continue shaking for 30 min.
2. If the packaging reaction (see “phage display- packaging T7” protocol) was stored with chloroform, spin the tube briefly in a microcentrifuge to separate out the chloroform. Leave the chloroform behind when pipetting the phage.
3. Based on the titer (see “phage display- tittering T7 protocol), calculate the amount of cells and phage needed. A ratio of 1E6 phage per 10 ml cells should be used. Mix the phage (packaging reaction) with the cells in a sterile 50 ml tube. This mixture must be plated within 20 min.
4. Transfer 1 ml aliquots of the phage/host mixture into sterile 15 ml tubes.
5. Add 10 ml molten top agarose at 45--50°C to each tube. Immediately pour the contents of the tube onto an LB plate with appropriate antibiotics (for BL21, just use plain LB). Spread the top agarose evenly by gently swirling the plate.
6. Allow the plates to sit undisturbed on a level surface so the top agarose can solidify. Invert the plates and incubate until the plaques are nearly confluent (3--4 h at 37°C or overnight at room temperature).
7. To elute the phage, cover each plate with 10 ml of Phage Extraction Buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 6 mM MgSO4) and place on a level surface at 4°C from 2 h to overnight.
8. Harvest the phage by tipping the plate slightly and pipetting the liquid into a sterile container. Combine the extract buffer from all the plates in a single tube or bottle. Add 0.5 ml chloroform and gently mix. Centrifuge at 3,000x g for 5 min to clarify the lysate and transfer the supernatant to a sterile tube or bottle. Determine the titer of the amplified library. Amplified library lysates can be stored at 4°C for several months without a loss of titer.
Amplifying fd-tet
1. Inoculate 100 mL of LB with 1 mL of K91 grown overnight in NZY.
2. Shake at 37 degrees until OD 1.0.
3. Slow the shaking down to 100 rpm for 10-15 min (to allow F-pili to regenerate).
4. Add around 1E10 TU of phage to the flask. If you do not have this much TU, then add as much as you can.
5. Slow shake (100 rpm) for another 15 min.
6. Pour culture into a pre-warmed 1L flask containing 900 mL NZY with .22 ug/mL tet. If the phage is expressing something from the tac promoter, for example, recombinant pVIII, then add IPTG to the flask. When I amplified the f88/6mer phage display library, I wanted a high density of recombinant pVIII on the coat of the phage, so I used 1mM IPTG.
7. shake at 250 rpm (at 37 degrees) for 35 min.
8. add 4 mL of 5 mg/mL tet stock to the flask (to make the culture around 20 ug/mL tet)
9. Take 20 uL of the culture for titering.
- make serial dilutions (in TBS) up to 1E-4.
- plate 100 uL of each dilution onto NZY/40 ug/mL tet plates.
- Invert plates and incubate at 37 degrees overnight.
- calculate the pre amplification titer based on the number of colonies on the plates. For example, if you get 20 colonies on the 1E-4 plate, then the pre-amplification titer in the flask was 2E6 TU/mL. If you are amplifying a library, then this titer represents the diversity of the library.
10. Continue shaking the flask overnight.
11. The next day, add kanamycin (to 50 ug/mL) to the flask. Allow it to shake for about another hour. This is to kill the cells. Extra cells in the phage stock can affect the titer and the diversity of a library over time. Kanamycin has worked well for me. I have also read protocols that recommend .02% sodium azide. Alternatively, you can purify the phage.
12. Centrifuge the amplified phage stock at maximum speed (4 krpm on the big centrifuge) for 30 min. This will spin down the cell debris.
13. Take the supernatant and store at 4 degrees. Fd-tet are stable for several months at 4 degrees, but just as a precaution, store some stock in 40-50 % (v/v) glycerol in the -20 degree fridge.
14. Spread 150 uL of the stock onto LB plates throughout the next couple weeks to ensure that the population of leftover k91 cells is approaching zero. My amplified f88/6mer library (which I added kanamycin to) originally showed 120 colonies of leftover K91 per 150 uL plated. Then in the next two weeks, the concentration of K91 went to zero.