I have a plasmid with the correct donor sequence. I want to use Crispr to insert it in frame . So in order to do that and lessen my cloning steps, I have to use Gibson Assembly for the insert plus the Homology arms. But before that i need too linearise it and my donor plasmid has some restriction sites 1kb upstream where i wanted it to be, so I am asking using proper primers can i delete such a big 1kbp piece??
As i understand in principle it can be done. But I am asking in reality has anyone done that before?
I wouldn't recommend trying to delete as much as 1 kb with Gibson. This would leave a lot of ways for things to go wrong, and you might not discover that they have gone wrong until you have wasted a lot of time. It would probably be easier to use PCR to amplify the sequence that you want, and then use Gibson to add your homology arms.
Or - if your desired sequence is not too long - just order a gBlock (etc.) and save yourself some steps that might cost more to undertake than the gBlock?
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