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lorenzo dep 03-09-2005 09:59 AM

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.
Anybody can help me sole this problem?

please write at [Only registered and activated users can see links. Click Here To Register...]
thank you very much

Lorenzo d'Episcopo
UNDERGRADUATE sudent Biotechnology


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