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RESEARCH DIVISION Laboratory Manual

 


 

Isolation of Phage DNA from Liquid Lysates and CsCl Block Gradients

Liquid Lysate (Genomic phage)

  1. Prepare an overnight culture of KW251 in 100ml LMM.
  2. Next day spin down 50ml of cells in sterile blue cap tube and resuspend in 25ml TM10 (10mM Tris HCl pH 7.5, 10mM MgCl2). Store at 4C.
  3. Add 50ul of cells to 5ml LMM and add one whole phage plaque (use a reasonable gauge pipette to get all of the plaque but avoid including more than one plaque). Include one tube uninoculated for comparison.
  4. Grow 37C until the cells are completely lysed and only bits are evident. This usually takes 4-5hrs. Do not overgrow or cells which are lysogenic for the phage will overgrow the culture.
  5. Store o/n at 4C. This stock can also be frozen at -70C as a high titre stock (ca. 106/ul) by adding an equal volume to 14% DMSO in TM10 (Make sure the DMSO is good quality).
  6. Next day add 6.5ml of 2X KW251 cells (step 2) to 100ml LMM and add 100ul phage stock from step 4 (a second one of 250ul is a useful backup in case 100ul is insufficient for lysis). Grow till complete lysed (usually 4-5hrs). To see lysis in the flask tilt the liquid and look through the top few mls against an overhead light. This makes it easier to see whether the remaining turbidity is due to bacterial debris or unlysed cells (the latter look "smokey or shinny"). Good lysis at this stage is critical to good yield. If lysis fails to occur repeat 100ml prep., halving the number of cells and doubling the phage added.
  7. Spin out the debris at 5-10K in GSA for 10min and then add s/n to a fresh GSA tube. Add RNAse A (preboiled) and low grade DNAse (ie not precious RNAse free grade!) each to 1ug/ml. Incubate at 37C for 20min.
  8. Add an equal volume (100ml) of 20%PEG 6000, 2M NaCl in TM10 (20g PEG 6000, 11.69g NaCl per 100ml).
  9. Chill on ice and then place at 4C o/n
  10. Spin 10K 10 minutes in GSA tubes and collect supernatant. Add 10g PEG 8000 to the supernatant and stir to dissolve. This step usually takes about 2 hours. (Phage can be left at 4C overnight at this stage).
  11. Spin 10K 10 minutes in GSA tubes and pour off supernatant, draining well
  12. Resuspend the phage gently in 4ml TM10 on ice for 1-2hr to allow to dissolve. Extract with an equal volume of chloroform (NOT phenol chloroform!) 1-2 times (Polypropylene tubes and spin 3.5K Beckman benchtop).
  13. Prepare a block gradient in SW41 tube with CsCl p1.4 (63g added to 100ml TM10) and p1.6 (104g added to 100ml TM10). Carefully add the layers with a autopipette. Mark with a texta the interface of the p1.4 and p1.6 as the phage band just above this.

  1. Spin 30K SW41 for 2.5hr. The band is a smokey white layer less than 1mm. Isolate band with a 18ga. needle IN A MINIMAL VOLUME.

DNA Isolation from the Gradient

  1. Phage in CsCl from a density gradient = 1 volumeAdd:1/10 vol 2M Tris pH8.51/20 vol 0.4M EDTA pH81 vol formamide
  2. 30' to several hrs room temp. Do not leave overnight or at less than room temperature.
  3. Add:
    - 1 vol H20
    - 6 vols EtoH
  4. Hook out pellet with spatula or spin 5 secs. Rinse in 70% EtOH and resuspend in 100-200ul TE over several hrs.
  5. Reprecipitate by adding 2.5vol. ethanol and NaCl to 0.1M final concentration to exchange Cs salt for Na salt. Resuspend in 100-200ul TE as above, allowing the long lambda DNA to go into solution (avoid long hard spins which tend to compact the DNA).
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998