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-   -   Cloning apparently worked but vector increased in size (http://www.molecularstation.com/forum/molecular-cloning-forum/83683-cloning-apparently-worked-but-vector-increased-size.html)

weenold 10-02-2011 01:14 PM

Cloning apparently worked but vector increased in size
 
Has anyone ever encountered this:

I cloned a 4.7kb insert into a 2.4kb vector. When I checked the transformants for insertion of the 4.7kb fragment by colony PCR I got a positive result but a few other smaller bands too. So I decided to mini-prep the plasmid and digest it. I found the 4.7kb insert but the vector showed up at about 3.5kb. Any idea what is going on? I used Novablue for cloning so the mutation rate should be low.

Thank you!

admin 10-04-2011 08:15 AM

Re: Cloning apparently worked but vector increased in size
 
Hi, there are several things that may have happened:

1) you cloned in as well something else (possibly a long pcr fragment/longer insert) as you mentioned the rate is low but its still possible

2) your plasmid is running higher due to a nick (incomplete digestion) - can you post your gel with ladder and controls - plasmid (original manufacturer tube) alone cut with same enzymes, and ligation (cloned plasmid and insert from colony isolation) cut with same enzymes.

Did you sequence both directions from the MCS? What was the result?


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