Hirt extract purification??

Victor Levenson levenson at uic.edu
Thu Dec 5 19:45:56 EST 1996


I have run into a problem with SV40-based shuttle vector and need some
input from those of you who used this technique.

I need to extract from COS cells (very small number: between 10 and
100) SV40-based vector and re-introduce it into E coli. My first
attempt was highly unsuccessful (no recombinants using DH5a with
efficiency of 2x10^9 on pUC)

I have prepared Hirt from 5x10^5 untransfected cells spiked with 100
cells containing the plasmid. ASfter precipitation with EtOH in the
presence of glycogen I washed precipitate twice with 70% EtOH, and
disolved in 10 ul H2O. Half of this amount was used for
transformation.

One of possible problems might be protein-SDS-RNA-etc in the
precipitate and I want  to purify plasmid before the next
transformation using one of those columns.  

Any input regarding transformation of E coli with Hirt-recovered
plasmid will be greatly appreciated.

Thanks,

Victor Levenson
Levenson at uic.edu




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