Baffling cloning results

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Aug 29 09:16:45 EST 2001


I checked the statagene catalog on this one.  You're gonna say D'oh!  Sure
cells are Kan resistant.  They contain the Tn5 transposon which has this
marker.

You'll either have to try different cells, or a vector with a different
marker, I think.

Mike

>Hi,
>
>Once again, it is time to turn to bmm-r experts.
>
>I am trying to clone a 2400-bp inverted repeat (1200+1200) into a
>13-kb vector (pBI121) carrying Kanamycin resistance.  I am using
>Stratagene's SURE cells (which worked for me with the same insert in
>pBluescript).
>
>Briefly:
>
>-- both insert and vector are cut with SacI and SmaI.
>-- Bands are eluted with Qiagen's QIAEX kit.
>-- Ligated with excess insert (~3:1).
>-- SURE cells transformed.
>-- Plated on LB-Kan (50 ug/ml Kan) plates.
>
>Result:  LAWN!
>
>When it happened the first time, I decided it was the Kanamycin.  Bought a
>new stock from Sigma (3 days in transit), made a fresh stock, sterilized
>using a 0.22um filter, re-poured plates making sure the LB-Agar was cool
>enough .... re-did the experiment -- with the same result.
>
>Will now troubleshoot ... but was looking for some insight.
>
>Thanks to all,
>Karthik


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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