Preparation of M13 RF DNA

Day 1
Streak out E. coli bacteria (usually strain JM103) on minimal + glucose plates and grow overnight.

Day 2
  1. Pick a single colony from the plate and grow for 7 hours in L Broth.
  2. Do serial ten-fold dilutions of an M13 bacteriophage stock to obtain a plate with approximately 200 infected colonies per plate. For each dilution, add 0.1 ml diluted bacteriophage to 10 µl of 10 mM IPTG, 25 µl of 4% Xgal, 0.2 ml of bacteria, and plate with 3 ml of 45°C pre-warmed top agar on a 100 mm Petri dish cont aining LB agar.
Day 3
  1. Remove an agar plug from the plate with a single blue (infected) colony on the top of it with the fine end of a sterile Pasteur pipet. Transfer it to 1 ml of L broth. Incubate while shaking at 37°C from 3 hours to all day (see note in #3 below).
  2. Inoculate 10 ml of L broth with uninfected bacteria from the minimal + glucose plate and grow to an OD550 = 0.5.
  3. Add the 10 ml of un infected bacteria (step 2) + 1 ml of the bacteriophage solution (step 1) to 500 ml of L broth and shake at 37°C from 4 - 7 hrs. (Note: Recombinant M13 grow at a slower rate than nonrecombinant vector. I have obtained increased yields by growing the bacteriophage in step 1 all day, and in step 3 overnight. For recombinant M13, increasing the ratio of infected to uninfected JM103 also increases the yield [ex. use 2 ml of infected bacteria from an all day culture + 1 ml (out of the 10 ml culture) of u ninfected bacteria and grow this mixture overnight. This should yield 1-2 mg of DNA/liter.)
  4. Centrifuge the bacteria for 10 minutes at 6,500 rpm.
  5. Save 1 ml of this supernatant for a single-stranded bacteriophage stock for future use.
  6. Resuspend the pellet in 100 mM NaCl; 50 mM Tris-HCl, pH 7.8; 10 mM EDTA. Centrifuge the bacteria for 10 min at 6,500 rpm.
  7. Discard the supernatant and freeze the pellet at -70°C for at least 2 hrs (overnight or longer i s fine).
Day 4 (Use this portion of the protocol to isolate M13 DNA, or a standard plasmid preparation protocol)
  1. Remove the frozen bacterial pellet and keep it in an ice bath for the following steps.
  2. Resuspend the bacteria in 15 ml of 25% sucrose in 50 mM Tris-HCl, pH 8.0. Bring to a final volume of 18.75 ml with sucrose solution.
  3. Add 3.75 ml of lysozyme solution (5 mg/ml in 50 mM Tris-HCl, pH 8.0; 10 mM EDTA) and gently rotate on ice for 5 min utes.
  4. Add 7.5 ml of 0.25 M Tris-HCl, pH 8.0, 0.25 mM EDTA and rotate gently for 5 minutes.
  5. Add 50 µl of RNAase (10 mg/ml stock) and rotate gently for 5 minutes.
  6. Add 30 ml of 50 mM Tris-HCl, pH8, 10 mM EDTA, 2% Triton X-100 and rotate gently for 15 minutes.
  7. Centrifuge at 12,000 rpm in Sorvall SS-34 for 20 minutes at 4°C.
  8. Remove and PEG precipitate the non-viscous supernatant with 1/2 volume 30% PEG6000, 1.5 M NaCl at 4°C for a minimum of 2 hrs (overnight is also fine).
Day 5
  1. Centrifuge the PEG precipitate at 3,000 rpm for 5 minutes at room temperature.
  2. Dissolve the pellet in 6.08 ml of 50 mM Tris-HCl, pH8; 50 mM NaCl; 5 mM EDTA.
  3. Dissolve 6 g CsCl to the above solution (technical grade is fine).
  4. Add 0.7 ml ethidium bromide (5 mg/ml) and adjust the refractive index to 1.390.
  5. Centrifuge in a small Oakridge tu be at 40,000 rpm for 40 hrs at 15°C.
Day 7
  1. Remove the bottom band (bacteriophage RF DNA).
  2. Add 0.4 ml of ethidium bromide (5 mg/ml stock) and transfer to an Oakridge tube with CsCl at the above refractive index. Centrifuge as above. (Although the 2nd centrifugation step will decrease the yield of RF DNA, it enhances its purity and, therefore, increases the probability that you will clone your insert rather than an unknown, bacterial DNA.)
Day 9
  1. Remove the bottom band (bacteriophage RF DNA).
  2. Extract the dye three times with an equal volume of n-butanol.
  3. Dialyze the DNA for 6 hours against 2L of 20 mM Tris-HCl, pH 7.5; 20 mM NaCl; 1 mM EDTA changing this solution twice.
  4. Ethanol precipitate the DNA and resuspend the pellet in TE or borate buffer for long-term storage.

RECIPES
Minimal Agar Plates (for 1L)
15 g Difco minimal agar (use Bacto Agar) in 800 ml H2O. Autoclave.
Salts: (makes a 5x solution. QS to 200 ml with H2O and autoclave)
10.5 g K2HPO4 (13.75 g for K2HPO4-3H2O)
4.5 g KH 2PO4
1.0 g (NH4)2SO4
0.5 g sodium citrate(2H2O)
Magnesium sulfate solution:
        Make and autoclave a 20g/100ml stock.
Thiamine-HCl Solution: (Various concentrations may be made )
        1% stock: 0.1 g thiamine-HCl (vitamin B1) to 10 ml water
             -or-
        20% stock: Dissolve 2 mg/ml water
        Autoclave.
Glucose Solution (20% stock):
        Dissolve 20 g glucose in w ater. QS to 100 ml. Add 10 ml.
Autoclave agar, salt solution, magnesium solution, and nutrients separately. Final concentration of D,L-amino acids is 40 µg/ml, or 20 µg/ml for L-amino acids. Vitamins are usually added to 1 µg/ml. These are then added to the salts after autoclaving. For our plates, we add 1 ml of the Mg stock solution, 25 µl of the 20% thiamine solution (or 0.5 ml of 1% stock), and 10 ml of glucose solution to the salt and agarose solutions. Other additives may be added as needed.
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
L Broth Agar
1 L of L Broth
15 g agar
Autoclave
L Broth Top Agar
Make L Broth Agar except substitute 7.5 g agar/L.
IPTG (100mM)
23.8 mg per ml autoclaved water.
Weigh out approximately this amount; note the amount weighed and dissolve in the appropriate volume of autoclaved water. Store at 4°C.
Xgal (4%)
50mg 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (Xgal) in 1.25 ml dimethylformamide.
Weight out approximately this amount, then determine the volume of solvent needed for final concentration. Light sensitive - Keep in fo il wrapped container. The solution should be clear. Discard if yellow-brown or pink.
0.1M NaCl; 50mM Tris-HCl pH 7.8; 10mM EDTA
20 ml 5 M NaCl
50 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0
Use HCl to pH to 7.8 (takes approximately 1 ml of concentrated acid).
QS to 1 liter with water and autoclave.
25% Sucrose in 50 mM Tris-HCl pH 8.0
25 ml 1 M Tris-HCl, pH 8.0
125 g sucr ose
Adjust pH with NaOH. QS to 500 ml with water and autoclave.
0.25 M Tris-HCl pH 8.0; 0,25 M EDTA
For 200 ml:
50 ml 1 M Tris-HCl, pH 8.0
100 ml 0.5 M EDTA pH 8.0
QS to 200 ml with water and autoclave.
RNAse (10 mg/ml)
Dissolve RNAse A in water. Boil for 5 minutes. Store at -20° C. Some protocols also add an equal volume of 40% glycerol and a half volume of 0.5 M NaCl.
50 mM Tris-HCl, pH8; 10 mM EDTA; 2% Triton X-100
For 250 ml:
12.5 ml 1 M Tris-HCl pH 8.0
5 ml 0.5 M EDTA pH 8.0
5 ml Triton-X 100 (v/v)
QS to 250 ml with water and autoclave.
30% PEG6000, 1.5 M NaCl
150 ml 5 M NaCl
150 g PEG (mw: 6000; mw = 8000 is OK to use)
QS to about 500 ml with water and autoclave to diss olve the PEG.
After autoclaving, QS as needed with autoclaved H2O to 500 ml.
50mM Tris-HCl, pH 8.0; 50mM NaCl; 5mM EDTA
For 200 ml:
10 ml 1 M Tris-HCl, pH 8.0
2 ml 5 M NaCl
2 ml 0.5 M EDTA, pH 8.0
QS to 200 ml and autoclave.
Ethidium Bromide Solution, 5 mg/ml
For 100 ml:
Dissolve 0.5 g of ethidium bromide with constant stirring in autoclaved water in sterile brown glass (or foil wrapped) bottle. This may take overnight to dissolve.
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Preparation of M13 RF DNA. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/m13rf.html). 1997. Oklahoma City. Revision Date: October 2, 1997."