problems with molecular cloning

Sergio yokese at hotmail.com
Wed Mar 9 13:04:06 EST 2005



lorenzo dep wrote:
> I have a problem an I need urgently some help.
> I am cloning a small peptide (300-400 bp) from a CS2 vector(4,1 kb)  
> into an Huc-int-GFP (about 13 kb)....
> What I should see are 2 bands: one for the plasmid (around 13 kb) and 
> the other for the fragment (between 300 and 400 bp).INSTEAD I see one 
> band around 2,5 kb and, in some cases, another band abotu 1,6 kb. I 
> never see any band which size correspond to the size of the sequence 
> that I should have cloned!

What is the marker of CS2 vector? ampicillin? what is the restriction 
map of CS2?... my bet is that you are picking colonies that have been 
transformed with the plasmid used as template. Getting rid of a 
supercoiled plasmid its not so easy even when gel-purifying the PCR 
product (I've seen it many many times).
If it happens to be the CS2 contamination, you may:
use a different selection marker of Huc-int-GFP, if possible.
use a smaller amount of CS2 for the PCR and digest the CS2 prior to PCR 
with an enzyme that cuts in several sites but not within the amplicon.

Good luck
Sergio



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