Protocol Online: Cached

Introduction

Lambda phage is a commonly used vector for transgenes. Its very high rate of infectivity makes it ideal for creating large numbers of clones. Large numbers of clones are often needed for construction of gene libraries that represent large populations of molecules. These libraries are also easy to sort through, as lambda phage clones are particularly easy to array spatially at high density. This is called plating a library. Here we will plate phage at various densities in order to generate a titer (count) and amplify a library in order isolate DNA.

This begins the second set in Series 2.

Set2

Phage Titering

Phage DNA

Gel Electrophresis

Plating Lambda Phage to Generate Plaques.
This procedure is used to isolate pure populations of phage from a single plaque or provide the titer of a phage stock. Serial dilutions are made of a phage lysate stock solution. In separate tubes, aliquots of each dilution are mixed with E. coli. Phage is allowed to adsorb to the cells and the cell/phage mixture is then heated to 37^oC, allowing the phage to inject their DNA into the cells. Top agar is added to each tube and the mixture is poured onto rich plates, which are incubated at 37^oC until plaques appear. Each plaque contains phages derived from a single infecting phage.

Here is an example of a dilution scheme, like one will use in this experiment in the dilution of the phage lysate.

The one below is a little simpler

Protocol

use sterile technique

1. Grow a culture of E. coli to saturation in NZY broth + 0.2% maltose. Done for you.

2. Melt top agar and maintain at 45^oC - 50^oC water bath. Done for you.

3. Add 100µl of E. coli culture to 5 Eppendorf tubes, labeled 1-5. Keep aside at room temperature to be used later for the infection with the diluted phage lysate.

4. Make serial dilutions of the phage lysate given in CSM. Each of these dilutions will be used to infect a separate sample of E. coli cells. Label 5 Eppendorf tubes,1-5, and add 90µLCSM to each. Add 10µL of phage lysate to tube #1, mix gently. Take 10µL from tube #1 and add to tube #2, and so on. You will now have five dilutions of the orginal phage lysate stock.

5. Add 10µl of one dilution of phage lysate, e.g. tube 1, to one tube (tube 1) of E. coli, 10µL of the next dilution (tube 2) to the next tube (tube 2) of E.coli, etc. Incubate at 37^oC for 30 min.

6. Label 4 NZY plates to correspond with labels on the dilution tubes.

7. Remove the tubes from the water bath. Add 100µLphage/cell mixture to 4 ml top agar, vortex lightly to mix, and pour the contents of tube onto a plate. Spread agar over the entire surface of the plate by tilting it gently.

8. Place the plates in a 37^oC incubator and incubate overnight.

9. Determine the concentration of the phage lysate in number of pfu/ml (pfu- plaque forming unit) from plates labelled 1- 4.

DNA Extraction Preparation:

Elution of Phage

Next Day or Session.

-Add 5 ml of CSM to plate # 1 and store at 4^oC for several hours with intermittent, gentle shaking.

-With a pasteur pipette, harvest as much of the CSM as possible and place in a 15 ml polypropylene tube.

-Add 1 ml of fresh CSM to the plate and store it for 15 minutes in a tilted position to allow all of the fluid to drain into one area. Again remove the CSM and combine it with the harvest.

-Add 0.1 ml of chloroform to the pooled CSM, vortex briefly, and centrifuge at 3000 rpm for 10 min at 4^oC. Recover the supernatant, add one drop of chloroform and store at 4^oC.

Go on to Phage DNA Extraction