problems with molecular cloning

lorenzo dep lorenzodep20 at hotmail.com
Wed Mar 9 04:59:07 EST 2005


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  lorenzodep20 at hotmail.com
thank you very much

Lorenzo d'Episcopo
UNDERGRADUATE sudent  Biotechnology


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