Baffling cloning results
kaghoram at unity.ncsu.edu
Wed Aug 29 08:34:06 EST 2001
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
-- 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.
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,
More information about the Methods