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RESEARCH DIVISION Laboratory Manual

 


 

Nested Deletions Using Exonuclease-III and Mung Bean Nuclease

ExoIII has a 3' to 5' exonuclease activity which must initiate on blunt ended DNA or DNA with a 5' overhang.

  1. A nested series of deletions can be produced by: cutting plasmid DNA (2.5ug per time point, see below) with an enzyme leaving a blunt (eg EcoRV) or 5' overhand (eg EcoRI) on the side that the deletions are to proceed. To prevent deletions in the other direction cut with an enzyme leaving a 3' overhang (eg. Kpn. NOTE: that SacII will not work for this purpose) or cutting with a 5' overhang creating enzyme and filling with thionucleiotides as follows. This is more reliable than using a 3' overhang:
    1. Cut with the enzyme to be filled in
    2. Heat 70 C for 15 min. Quench on ice.
    3. Add thio-d NTP to 40uM and 5U klenow
    4. 20 min at RT. Heat to 65C for 10min.
    5. Phenol/CHCL3 extract and ethanol ppt.
    6. Cut with the second enzyme on the side that deletions are to proceed in around 100ul. Heat inactivate the enzyme when complete. A linearized clone and linearized vector (1ug ea.) provide useful markers for the start and end of the deletion series, respectively, when the deletions are run on a gel.
  2. Resuspend the DNA in
    - 1X exo buffer (see end)
    - 10mM 2-mercaptomethanol (prepared fresh from 14M stock)
    - Xul DDW
    - The volume should equal 25ul per time point. Heat to 30 for 5 min to equilibrate at incubation temp.
    - Dispense 20ul 10X mung bean buffer, 155ul DDW into eppendorf tubes for each time point.
    - Have on hand crushed dry ice.
  3. Add 20U of ExoIII per ug of DNAand vortex.
  4. Remove 25ul aliquot’s into the diluted mung bean buffer at Xsec intervals according to the following table and place on dry ice.
    - 37 = 400bp/min
    - 34 = 375bp/min
    - 30 = 230bp/min
    - 23 = 125bp0/min
       

    -

    I generally do my incubations at 30C and take my first aliquot at 20 sec and thereafter at 40-80 sec intervals (a test for the best timepoints may be useful).
  5. When finished remove tubes from dry ice and heat to 68C for 15 min then place on ice.
  6. To make the DNA blunt ended, add 7.5U mung bean nuclease (diluted in mung bean dilution buffer, see end) per 2.5ug time point and incubate at 30 for 30 min.
  7. Ethanol ppte, resuspend and load onto a 0.8% TAE gel. Run and excise bands free from undigested DNA. Isolate DNA and ligate overnight. Transform and pick 2-4 colonies per time point for mini gel analysis and subsequent sequencing.

 

Buffers

- 2X exo buffer
- 100mM Tris pH8
- 10mM MgC12
- 20ug/ml tRNA
- 10X mung bean buffer
- 300mM NaAC pH 5
- 500mM NaCl
- 10mM ZnC12
- 50% glycerol
- 1X mung bean dilution buffer
- 10mM NaAC pH 5
- 0.1 mM ZnC12
- 1mM cysteine
- 0.1% triton X-100
- 50% glycerol

Comments:

  1. The DNA must be >85% supercoiled. ExoIII will digest from nicks. Use CsCl purified DNA and avoid using the UV lamp to isolate plasmids from CsCl gradients. Check the DNA on a gel vs. cut plasmid.
  2. The DNA must be fully restricted with the 5' overhang (otherwise background of undigested DNA) and with the 3' overhang (otherwise deletions proceeds in both directions).
  3. Use a gel to isolate the deletion fragments rather than simply extracting the DNA (Extraction of the deletions after the mung bean step requires the addition of 4ul 20%SDS, 10ul 1M Tris pH 9.5, 20ul 8M LiCl and 250ul phenol/CHC13, followed by re-extraction and precipitation) after the mung bean step. Gel purification is preferable as it removes the background and allows you to monitor how well the reaction has proceeded.

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