"For the linear transfer, I am scared if the exonucleases would just chew of the construct. Is there any literature on how to avoid that, also won't the low temperature in ice bath inactivate the enzymes?
Thanks,
Harsh"
Yes, exonuclease activity is required for recombination to occur. The viral beta protein will protect the single stranded fragment and promote insertion of the construct as an okazaki fragment. Here is a good recombineering guide from Stewart's lab.
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