Promoter for Ampicillin in plasmids.

h9290184 at hkucc.hku.hk h9290184 at hkucc.hku.hk
Sun Mar 20 05:28:57 EST 1994


Hi netters,

I am currently trying to insert a putative promoter fragment in front of
the LacZ gene for promoter activity assay. The fragment in question is around
15 Kb or so. The problem is that the few transformants I get have deleted the
LacZ gene while taking up the 15 Kb fragment. The LacZ gene itself was
subcloned into pBluescript with some difficulty so I suspect that the deletion
arises from the duplication of the partial LacZ sequence in the construct. I
think I can remedy that by removing the LacZ and F1 origin of replication by
cutting with SacII and (partial) SspI followed by bunt-ending and religating.
 
My question is would I be disabling the promoter used for the Amp. gene by 
 cutting with SspI?
I plan to do the same with a construct in pUC18 by removing the HindIII-SspI
fragment. From the reference I get from the NEB catalog page 158, the mature
beta-lactamase is from 2417 to 1629 while the SspI site is at 2501. I do think
that both plasmids share this same region if not more (from base 756 to 2686 by
fasta on linear sequences).

Thanks x10^6.

Kaimin Chan
Institute of Molecular Biology
The University of Hong Kong
Hong Kong
email:h9290184 at hkucc.hku.hk 



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