Dear all,
I'm using a lentiviral vector backbone from abm and have issues setting up double digestion. I observed the un-cut plasmid which is 9.2 kb is about 10-11 kb when run on a gel and has no band after digestion. Can I still use this plasmid to perform restriction digestions and subsequent transductions?
Thanks, Ranjitha!
Could you indicate which cells you used for cloning. lentiviral vectors are usually cloned in strain Stbl3, which happens to be endA+, therefore explaining why you got no band after the digestion. Also I would suggest you prepare DNA from multiple clones if you are not sure since these kind of vectors are prone to recombination which could give improper constructs with altered sizes. and finally both forms of the plasmid should be digestible.
Hanna Alalam thanks for the suggestion!
I used One Shot Stbl3 competent cells for transformation and performed an additional wash step during plasmid prep. I'll check out if the plasmids from other clones are the same.
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