need help with subcloning !! - (Dec/21/2011 )

Hi

I am trying to clone a 350bp fragment from a pbabe into a plenti6 with N_terminal flag ha ( 6500bp). For this, I cut out the fragment with BamHI/EcoRI and I digest the plenti with BamHI/EcoRI as well. I obtain the fragment on gel and extract the band using machery_nagel gel extraction kit, purify it. At the same time, I also obtain the digested plenti vector of the correct size. I ligate them using T4 DNA ligase (fermentas) overnight at 4 degrees and transform 100µl of the competent XL1 bacteria. For ligation, the vector : insert = 1:3 molar ratio and i take 100ng of vector and accordingly the insert.

Problem 1: I do get a lot of colonies on my main plates but a lot more on the vector only plates.

Problem 2 : however, i picked some colonies and did minipreps, did a Bam/Eco control digest to check if the ligation had worked. i get 6 out of 24 colonies positive i.e. I have the 350 bp fragment and the digested vector at 6500bp.I sequenced 4 of these 6 clones to do a final check if the fragment was correctly inserted. Now this is the point, where i am a bit lost!!

clone 1 : the desired fragment is present with the RE sites but there is a stop codon just after the flag ha tag in the correct ORF. I dont get it ??

clone2 and 3: the desired fragment is present in the correct ORF with the n-terminal flag ha tag but I cant find the site for ecorI, instead just after the stop TAG, there is GAATTTT in place of the usual GAATTC eco site. and this GAATTTT is actually present somewhere later in the 3'region of the plenti backbone, which means that my ligated vector is missing a part of the 3' region. The rest of the 3'backbone is fine. but the missing part had the blasticidn resistance gene, without which this plasmid is useless for me.

I would welcome any suggestions to improve my cloning strategy. Is there some star activity exhibited by EcoRI or plasmid recombination ?? How can I reduce the star activity of ecoRI if that is the case. Should I use some other bacteria or bacteria freshly made competent ?

Thanks a lot for your replies!!
Jas

????????????? no body can help me !!!

It sounds as if your ligations are working, but that the construct you are making is toxic to the cells. This leads to selection for mutations that remove the toxicity, which I think is what you are seeing. You may have trouble making the construct you are looking for. Things you can do include moving to a lower copy number vector and growing at a lower temperature, but there are things that simply won't clone.