I need help: Subcloning

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Thu Sep 24 08:33:55 EST 1998


"Patrick F.H. Lai the Graduate Student" <pfhlai at LOOKSMART.COM> wrote:
[DNA containing a PCR product subcloned by TA cloning cannot be cut with AatII
 and BspDI:]

> Sequencing indicated that the insert is perfect, with the AatII & BspDI sites
> in there as expected.  ( I was so happy. )
> 
> I proceeded to digest the MiniPrep with AatII & BspDI, hoping to gel extract
> the released fragment for ligation into the incomplete cDNA.  But i have
> tried it 5 times and my ~0.6 kb fragment is nowhere in the agarose gel to be
> seen !!!!?????

[...]

> the bacteria producing modified DNA ?
> Well, I tried minipreps from JM109 and TOP10.  Same results.
> These strains are not known to modify plasmid DNA anyways.   Right ?

JM109 is not dam- . I don't know about TOP10 for sure, but the information
I have indicates that TOP10 is not dam- either. BspDI does not cut dam-
methylated DNA, i.e. if your restriction site appears in the context
AT!CGATc it won't be cut if propagated in a dam+ strain.

Therefore your best bet is to transform your Miniprep DNA in some dam-
strain, like JM110, isolate DNA, cut out your insert and be happy.

(PCR products are obviously unmethylated, therefore your enzyme cut there
just fine.)

BTW, you could have found this out yourself by looking into the NEB catalogue.

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */



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