DNA isolation from phage lysates (Qiagen)

The Phage Cookbook by Dominik Refardt

DNA isolation from phage lysates using the Qiagen lambda midi kit

(as far as I know, the kit is not produced anymore)

The protocol here was developed to obtain phage from induction of liquid cultures. If possible, do a plate lysis (here, this was not possible).

• 1. Inoculate 5 ml LB with a single lyosgenic colony and incubate overnight.

  2. Dilute the overnight culture 1/100 in 25-50 ml LB and incubate at 37 °C with vigorous shaking for 3 hours.

  3. When the culture reaches a density between 2·10⁸ and 5·10⁸ cfu/ml (OD₆₀₀ = 0.2-0.4), add 5 µg/ml mitomycin C and incubate for 2 hours. Add 2% v/v chloroform and continue incubation for another 15 minutes.

  4. Follow instructions of the kit. Don't forget to add MgSO₄ (final concentration 10 mM) to the lysate before the DNase treatment!

If possible, try to obtain lots of free phage without mitomycin C induction, because one needs a hell of a lot of mitomycin C.

It is important to induce the culture before it is too dense. If the culture approaches stationary phase, the phage titer begins to drop (figure 2.5.1). This is somewhat contradictory to figure 2.6.2, I don't know why.

Table 1: Results that were obtained using the Qiagen lambda midi kit