freeze-squeeze

Eitan Rubin bcrubin at dapsas1.weizmann.ac.il
Tue Mar 5 03:59:18 EST 1996


I am using a much simpler protocol for purifincation of PCR amplifiable DNA:

1. Cut the band out (I use simple TBE gels)

2. Freeze in Liq. N2

3. Spin 5min at top speed in a table top cetrifuge (eppendorf)

4. Remove aqueous layer (top) to fresh tube.  Be sure to leave any 
debris behind.

5. Add 200uL ddw, and repeat the freezing and centrifugation in steps 2-3.
Collect aquaous phase into the same tube.

6. 2uL of this phase are sufficient for PCR even if the original band was
vary week. Do not use more.

   Eitan.


In article <DnK0zp.L1A at pfizer.com>, "Mark R. Miglarese"
<miglaresemr at pfizer.com> wrote:

> Here's a reference for phenol freeze isolation of DNA from agarose:
> 
> Bewsey et al., 1991.  Biotechniques 10:724-725.
> 
> 1.  Cut the band out (use FMC SeaKem GTG agarose), push it through a 1 
> cc syringe with 25 g needle into a microfuge tube.
> 
> 2.  Add 20 uL 3M NaOAc pH 5.2 plus 2 volumes (about 300-400 uL) tris- 
> buffered phenol and vortex vigorously.
> 
> 3.  Freeze on dry ice, spin for 5 min at room temp
> 
> 4.  Remove aqueous layer (top) to fresh tube.  Be sure to leave any 
> debris behind.
> 
> 5.  Extract with 1 volume phenol: CHCl3 (1:1) twice.
> 
> 6.  Extract once with CHCl3.
> 
> 7.  EtOH ppt and wash pellet (may not be visible) with cold 70% EtOH.
> 
> 8.  Resuspend DNA in appropriate volume of H2O.
> 
> 9.  Quantitate and ligate, label, etc.
> 
> This method has served me very well.  EtBr and BPB have never been a 
> problem for me (even with frags comigrating with BPB).  Good luck.
> 
> Mark R. Miglarese, Ph.D.
> Cancer Research
> Pfizer Central Research
> Box 372                        
> Eastern Point Road              
> Groton, CT 06340
>                 
> Phone: (860) 441-8478
> FAX:   (860) 441-1290
> Email: miglaresemr at pfizer.com
>

-- 
Eitan Rubin, Plant Genetics, Weizmann Inst. of Science
Phone: (972)-8-9342421
Email: bcrubin at dapsas1.weizmann.ac.il
                  Shalom Haverim



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