HELP! MY construct (A different one!) is making me crazy!

Heidi Moss hmmoss at MAIL.MED.CORNELL.EDU
Thu Jan 26 16:36:31 EST 1995


I am trying to ligate 2 human genomic fragments (one is EcoR1/SalI the
other is XbaI/SacI) into pBluescript SK+ that
are 6kb and 7kb respectively. To make a long story short, I have never
obtained positive clones. In terms of troubleshooting, this is what I
have done:
1) Permutated the vector:insert molar ratio in various ways (isn't 1:3
around the optimum?)
2) Switched E-coli hosts from XL1-Blue to Sure 2 (I had some data that
the XL-1 Blue was deleting/rearranging some of my gene)
3) Tested ligase/buffer activity and it was OK
4) Ran an agarose gel of vector alone--insert alone--ligation reaction
and hybridized: it seems that the ligation reaction was somehow
sub-optimal: the ligation band was fairly strong, but there were also
other bands/smear (that run BETWEEN the ligation band and insert band) in
addition to VERY FAINT unligated insert and vector. What are 
these bands/smears?
5) Varied transformation conditions (ie/ amt of ligation 
reaction added)
6) My mini preps always look weird -- many bands, most clones 
were different from each other, some vector size, some shifted vector size, 
some nowhere near the vector size, ALL with weird insert/vector 
conformation sizes.
(digested appeared DIFFERENT than the undigested, and hybridization data
showed that none of the clones were what I wanted. What the heck was I
cloning??)

Is there a better vector than pBluescript that I could try for these 
inserts? Do you think I still have a host problem? What about insert
preparation? ( I read the TIBS article, and for me, a gentle GENECLEAN prep
seems best). I have cloned smaller inserts easily in the past, but for these I
feel like I have tried and read everything to no avail. 
Although I am now shooting for smaller insert subsets to clone, I'd still 
like to solve this mystery. Any advice would be appreciated!!
MANY THANKS!!
-- Heidi
               






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