band separation prob. on agarose gels

Kyle Legate legatek at hotmail.com
Mon Feb 23 13:03:47 EST 2004


Ferdinando Pucci wrote:
> On Mon, 16 Feb 2004 18:14:31 GMT
> "Kyle Legate" <legatek at hotmail.com> wrote:
>
>> I'm trying to do a simple sticky-sticky cloning step using EcoRI and
>> NotI. I cut 5 microg. of each plasmid. One plasmid yields 4.3kb and
>> 2.5kb bands, and the other plasmid yields 3.5kb and 1.6 kb bands. I
>> digest both plasmids and separate the bands on a 1% agarose gel in
>> TAE at 5V/cm. I
>
> Did you inactivate the enzymes? How many wells did you used?
>
I used 2 wells joined together to accomodate that amount of DNA, and I've
never found a reason to inactivate my enzymes, since I never detect activity
after gel purification.





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