Baffling cloning results

Karthik Aghoram kaghoram at unity.ncsu.edu
Wed Aug 29 08:34:06 EST 2001


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




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