problems with molecular cloning

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).
First I run a PCR to amplify the sequence encoding my fragments and to
introduce the restriction sites for XhOI. I check the PCR products on a
gel, I digest the fragments and the vector Huc-int-GFP with XhOI, I
dephosphorilate the vector with "Shrimp alkalyne phosphatase " and I run a
gel with the fragments and the vector. Then I perform a DNA purification
from the gel (from the right bands) and I run 1 uL on a gel to compare the
relative concentrations of the fragments and the vector.
I do a ligation, I transform E.Coli cells (DH5a),I plate them in petri
disches prepared with LB medium and Ampicilline. After one night at 37 C I
make a "colony PCR" to check which colonies really contain the fragment of
interest and then I chose the positive clones to grow a miniculture in LB
medium and Ampicilline.
Everything "seems" to be fine untill this point.
BUT after all this I make an extraction of the plasmid DNA from the
miniculture, I cut again with XhOI to check if I really have the fragments
that I am cloning.
I run a gel with the digestion product.
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!
Anybody can help me solve this problem?

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thank you very much

Lorenzo d'Episcopo
UNDERGRADUATE sudent Biotechnology


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lorenzo dep wrote:

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