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
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.
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
Email: bcrubin at dapsas1.weizmann.ac.il
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