Method: Growth and Purification of 25-100 ug Lambda Clone DNA

June 26, 1990

C. Helms

Principle:

There are essentially four parts to this protocol:

1. growth of at least 5x10e8 pfu phage to provide an inoculum growth of a larger liquid lysate that will produce about 5x10e12 pfu
2. concentration and purification of the phage, and
3. DNA preparation.

The protocol is presented as it would be performed on five consecutive days, total time about 5 hours.

1. LMM
2. Large-scale growth medium
3. 20% PEG-8000, 2.5 M NaCl

Growth of liquid lysate for inoculum:

Day 1

1. Start a 5 ml overnight culture of LE392 in LB containing 1mM Mg++ and 0.2% maltose (LMM).

1. Streak out the phage that you wish to grow for single plaques using the LE392 overnight.
2. Start an overnight culture of LE392 in LMM. There should be about 0.3 ml of this culture for each phage that you plan to grow.

1. Pick a single plaque from each streak and add it to 0.5 ml of SM+ or lambda diluent +. Allow the phage to elute for at least 15 minutes at room temperature. Use a 15 ml polypropylene culture tube.
2. Prepare a 1:6 dilution of the LE392 overnight culture in LB medium. Prepare enough of the dilution to provide at least 0.2 ml for each phage to be grown. Be sure to save enough of the undiluted culture for the titration step (about 0.2 ml for each different phage).
3. Add 0.2 ml of the diluted overnight culture to each plaque eluate and allow the phage to adsorb for 15-20 minutes at room temperature.
4. Add 5 ml of LB and incubate the infected culture on the 37 degrees C roller drum. Lysis of the cultures occurs at about 3 1/2 to 4 hours so they should be checked frequently after about 3 hours to ensure that the lysate is harvested as soon as lysis is complete.
5. When all of the cultures have lysed, spin at 8,000 rpm for 20 minutes to pellet the bacterial debris. Transfer the lysate to a new tube for storage. (add a couple of drops of chloroform to the tube for long term storage)
6. Titer the lysates by standard methods. Expect the titer to be between 2x10e9 and 2x 10e10 pfu/ml. Use the remainder of the LE392 culture in titering.
7. Growth of a larger liquid lysate:
   1. Start an overnight culture of LE392 in LB medium. There should be about 3 ml for each different phage.

Day 4

1. Count the titer plates, calculate the titer and the volume of each lysate containing 5x10e8 pfu.
2. For each lysate, mix 2.5 ml of the saturated LE392 overnight and 5x10e8 pfu in a large test tube. Allow the phage to adsorb for 20 minutes at room temperature (or 15 minutes at 37 degrees C.
3. While the phages are adsorbing, prepare the large scale growth medium. Distribute 50 ml of this broth into a 250 ml flask for each lysate to be grown. Be precise in the measurement of this 50 ml since the volume is important in subsequent steps.
4. Add the infected cells to the flasks and incubate with vigorous shaking at 37 degrees C for about four hours. The cultures may not lyse completely, but there should be evident clumps of lysed cells which may appear like bits of fine glass wool. (If lysate hasn't cleared after 6 hours recheck the titer and start over).
5. Add 0.1 ml of chloroform, shake the culture vigorously and then let the chloroform settle.
6. Add DNaseI and RNase at 1 ug/ml each. (These solutions are conveniently stored as 1mg/ml stocks in glycerol containing buffer at -20 degrees C and added as 1 or 2 drops per culture. ) Tilt the culture gently to mix in the enzymes without redispersing the chloroform. Incubate at room temperature for about 20 minutes.
7. Split the lysate into equal halves in two 37 ml plastic tubes and centrifuge out the bacterial debris for 10 minutes at 10,000 rpm.
8. Save 10 ul of the supernatant from each phage in an eppendorf tube. (This small aliquot may later be analyzed by the quick lambda DNA check procedure to ensure that there is enough DNA to continue with the subsequent purification and DNA isolation steps.)
9. Decant the remainder of the supernatants directly into new 37 ml plastic centrifuge tubes.
10. Phage concentration and purification:
    • Add 7 ml of a solution of 20% (w/v) PEG-8000 and 2.5M NaCl to each centrifuge tube. Mix well and store overnight on ice (0 degrees C) in the cold room.

1. Centrifuge the PEG-precipitations for 15 minutes at 8000 rpm in an SS34 rotor. Decant the supernatants and then invert the tubes over paper toweling for 5 to 10 minutes to drain. Do not allow the pellet to dry out!
2. Resuspend each pellet in 1 ml lambda diluent. Rinse the sides of the tubes well since the precipitate often is stuck to the side rather than at the bottom of the tube.
3. Pool the resuspensions for each phage in one of the 37 ml tubes. Transfer to solution to a polypropylene tube and add 2 ml of chloroform (do not use chloroform with polycarbonate tubes). Centrifuge at 10,000 rpm for 10 minutes.
4. Transfer the aqueous phase to two eppendorf tubes and perform two additional chloroform extractions with 0.5 ml of chloroform per tube.
5. Combine the aqueous phases and add an equal volume (~ 2 ml) of sterile water or 10 mM Tris-HCl.

1. The sample may now be loaded directly onto a standard 2 ml DE52 column, the phage purified and DNA extracted as described for a standard lysate, except that it is not necessary to add any carrier (i. e. mussel glycogen) to the isopropanol precipitation and the DNA must be resuspended in an appropriately larger volume of TE (e. g. 40 - 120 ul). The average final yield is about 60 ug.

1. LMM medium (LB containing 1mM MgCl2, and 0.2% maltose)

   
   	Use sterile ingredients:
   	150 ml bottle of LB
   	1 ml 20% maltose
   
   

2. Large scale growth medium

   
   	Use sterile ingredients:
   	150 ml bottle of LB 
   	300 ul of 1M CaCl2
   	375 ul of 40% glucose
   
   

3. DNaseI stock solution

   
   	Prepare a 1 mg/ml solution in 40% glycerol 
   	containing 150 mM NaCl.  
   	Store at -20 degrees C
   
   

4. RNase stock solution

   
   	Prepare a 10 mg/ml solution 
   	in 10 mM Tris-pH7.5, 
   	15 mM NaCl. Heat to 100 degrees C 
   	for 15 minutes to destroy nucleases. 
   	Allow to cool slowly to room temperature. 
   	Add an equal volume of sterile 80% glycerol.   
   	Store at -20 degrees C.
   
   

5. 20% (w/v) PEG-8000 and 2.5M NaCl

   
   	100 ml sterile 3M NaCl 
   	25g PEG-8000 
   	Mix, with heat if necessary.  
   	Adjust the volume to 125 ml.
   
   

References:

Helms, C., Dutchik, J.E., and M.V. Olson. (1987). "A lambda DNA protocol based on purification of phage on DEAE cellulose". In: Meth. Enzymol., 153: p 69-82. Eds.: R. Wu, L. Grossman. Academic Press.

CRI Laboratory Manual: RFLPs Project (1989).