cosmid/genomic DNA ligation problem

Hiranya S. Roychowdhury hroychow at nmsu.edu
Tue May 30 16:47:58 EST 2000


Yes... I am sorry I mentioned direct trasformation. Rodney's post sounded as
if he was trying to do direct transformation of E. coli, which is also
possible with cosmids (although not the most efficient and defeats the purpose).


At 11:48 AM 5/30/00 -0600, Warren Gallin wrote:
>    The whole point of using a cosmid is to package the ligation product using
>a lambda packaging reaction a) to make the transformation go well and b) to
>select for insert sizes that are large enough to be effectively packaged into a
>lambda phage particle.  Therefore, the goal is to produce long concatemers, not
>single closed circular recombinants, as you would with a plasmid.  Assuming
>that you are using dephosphorylated cosmid, you would want a much higher vector
>to insert ratio than you would use for a plasmid ligation.  The desired
>ligation product would be way up at the top of the gel; it wouldn't enter most
>agarose gels.  You certainly shouldn't be doing a gel purification on your
>ligation.
>
>    Without a clearer statement of your ligation and packaging conditions,
>though, it is hard to get more specific.
>
>Warren Gallin
>
>"Hiranya S. Roychowdhury" wrote:
>
>> You need to elaborate on the protocols you are using.
>>
>> In general, while there is a practice of running out a portion of the
>> ligation rxn out on a gel, I have not put much stock on it. To me, the proof
>> is in the pudding. Ligation reactions are not that tricky (if the DNA are
>> reasonably clean), it is the transformation of bacterial cells that get us
>> most of the time (especially with larger plasmids). I would suggest using an
>> efficient transformation protocol, eg. Hanahan's fresh competent cell
>> method. It has worked for me for plasmids upto 20kb, but then your cosmid
>> clones may be even larger.
>> Electroporation may work even better, but if used for library construction
>> one should do multiple sets.
>>
>> At 10:28 AM 5/30/00 -0500, Rodney Earl Pettway wrote:
>> >I did a cosmid/genomic DNA ligation reaction and ran some of the
>> >ligation out on a gel.  I have never ran a ligation out on a gel before
>> >so I am not sure if it worked or not. I carried out my transfection and
>> >plated out the bacteria and I did not get any colonies.  Could it be
>> >that my ligation did not work or something else.  If it is my ligation
>> >reaction what could I do to solve this problem?
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Dr. Hiranya Sankar Roychowdhury
GENE LAB/ EPPWS
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu


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