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Cloning 4.2kb insert in 29.8 fosmid

Cloning 4.2kb insert in 29.8 fosmid - Protocols and Methods Forum

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Old 11-28-2003, 03:58 PM
onels@bluemail.ch
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Default Cloning 4.2kb insert in 29.8 fosmid



Hi all,
It is weeks that I am trying to obtain a good contruction, I first did ligations with these agarose
gel purified fragments, then I set up different ligation with different molecular ratio 3:1, 1:1, 1:3
and I transfected E. Coli. The transfection was not very efficient but I obtain several nice colonies
with all ratios and the control (cutted fosmid alone) was good (no colonies).
But once I do minipreps and digestion controls, my construct is much more shorter than what I
expect and not allways the same (8kb to 25kb). I think that there is some deletions that occur
within bacteria and I tried different bacteria (DH5alpha heatshock, DH10 electro, XL1blue
heatshock) but I always have the same problem. Is my insert toxic? Do you know some bacteria
strain wich is more appropriate fore such huge construct?
I would be very grateful if someone could give me some goog advices!
Kind regards.
Paul


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