purification of a 140 kb fragment

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Sun Mar 3 08:03:40 EST 1996


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From:  Andrei Popov <ANDREI.POPOV at bbsrc.ac.uk>
Message-ID:  <0548441402031996/A20727/IAPC/11A313883300*@MHS>
To:  methods at daresbury.ac.uk (IPM Return Requested)
Subject:  RE: purification of 140 kbp from agarose
Importance:  High
Sensitivity:  Company-Confidential

: Hi,
:       lately I have been trying to isolate a pulse-field gel
: fragment from agarose. GeneClean didn't work at all so I went on
: with an agarase digestion of the agarose followed by a dialysis
: over a millipore membrane sitting on a becher full of TE. This
: dialysis usually work very well for smaller fragment. But after
: this step, I precipitate with 1/10 volume of 3M NaoAc and 3 volume
: of cold 95% Ethanol, rinse with 80% ethanol and resuspend in 10 ul
: of dH2O, run on a gel and there is nothing. I even hybridised it to
: see if there was a trace amount. But there was nothing. I also tried
: a phenol extraction with no dialysis followed by the precipitation,
: and still nothing. I also tried this thing called GeneCapsule, which
: uses electrophoresis to get the DNA out of the agarose and onto a
: membrane. But the after an overnight run at 100 volts, the DNA was
: still in the plug.?????. So if any of you out there have any idea
: please share them with me.
: Many thanks
: Ghis
: P.S. I used low-melt agarose, but my pulse-field gel would be much
: nicer if I could use a non-low-melt agarose.                   

Hi Ghis,
you described a standard protocol that should work.
Here are some suggestions:
1. make plugs with as high DNA concentration as practicaly possible
2. Dr. A.Schedl et. al (see NAR 21:4783) used to run normal preparative
   gel than another one at 90 degr angle to concentrate DNA in 4% LMA agarose.
   This gives you 5-fold concentration of your sample.
3. I missed the point of membrane dialysis. Normally people do it when
   they want to microinject large DNA w/o precipitation
4. If the amount of DNA after agarase digestion is really low 
   try co-precipitation with glycogen
5. If the fragment is a YAC, beware that you co-purify some amount of the 
   2-mu plasmid (which has multimeric forms)- it can interfere with some
   applications (probe)
6. check the presense of DNA after agarase- some batches are no good at all,
   or the buffer might be contaminated with nucleases
7. You did not specify your final goal, but sometimes LMA does not interfere
   with further enzymatic protocols (Klenow, REs, T4 ligase etc...)
   Of course there is no need to stain ALL the gel with EthBr- only
   outer lanes. Having your DNA in agarose even has some advantages-
   you can change the buffer by washing the block 

good luck

Andrei Popov






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