Basic Subcloning Question

[David L. Haviland, Ph.D.]

At 11:03 6/4/99 -0400, Nicolas Lemee wrote:

For the most part, I can agree with what is said here...

>I totally agree on the lab technique here regarding CIP (I don't know
about 'farting
>around' all day...). Ratios (and quality: always geneclean and/or etoh ppt
before you
>ligate) of insert/vector during ligation is a better way to go at it. I
usually
>guestimate the ligation at 1 vector:3insert overnight at room temp, zap
and plate DH5a
>from gibco in the evening next day (expensive but you get your clone every
time,

With exception of this...

> I always wonder at people making their crappy home grown comp. cells and
then using
>qiagen columns to miniprep the plasmids out) . 

If one has access to an electroporator, I wonder about those that insist on
buying electrocomps!   Given how *easy* they are to make AND *match* the
transformation effiency of store-bought cells, why buy them?  Save the lab
$$ for more important items like enzymes.

>I then miniprep 8 to 18 colonies
>(depending on how good  looking the plates are). The boiling minpreps take
less than
>1h15 total time, 1 hour digest , 45 minutes gel run: in one morning you
got your
>answer, works every time !

I'll agree here... although we've had good luck with Wizzards, when they
fail, we always go back to the "tried and true" home-grown alkaline lysis
minis/boiling minis/PEG minis - as long as it sequences.

David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
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