How big DNA fragment can I clone to pUC19 plasmid? (396-452 multiple cloning site (EcoRI-HindIII)) Is there any general rules concerning the size of an insert versus the size of a plasmid?
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reijokin
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Posted 11 March 2005 - 08:35 AM
I have to answer this to myself. I got an answer from Aldevron that their scientists recall that there are a paper that says that 19 kb insert is successfully inserted into pUC19. They weren't sure. But generally speaking, it's about 10 kb that is recommended to be the limit for fragment size used in plasmid cloning.
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sergechampetier
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Posted 14 March 2005 - 05:54 AM
I have once cloned a 15-kb fragment, and someone told me she cloned a fragment more than 20kb into a usual plasmid.
One very significant factor for large inserts is the method of purification. Using kits based on sodium iodide (e.g. Qiagen) are OK for normal-sized fragments (1-4kb, say) but for really large pieces I recall a phenol-chloroform-based extraction yielded the best results.
Good luck.
One very significant factor for large inserts is the method of purification. Using kits based on sodium iodide (e.g. Qiagen) are OK for normal-sized fragments (1-4kb, say) but for really large pieces I recall a phenol-chloroform-based extraction yielded the best results.
Good luck.
