Hi everyone,
This is my first time using the Q5 kit.
I‘m trying to insert 200 nucleotides into my construct.
Reading up online, I see that only 100 nucleotides are tolerated at a time so I’m thinking of inserting ~60 nucleotides at a time for a total of 3 reactions.
Would anyone know if it’s necessary to go through the entire amplification/KLD/transformation process after each insertion or can I just use the PCR product and do the rest of the insertions right after?
Thank you in advance!
what is the size of your plasmid? if it less than ~6kb, you could try neb HIFI.
just PCR your plasmid and pcr you ~200bp insert with over laping ~20-40bp.
Yeah I agree with Junjie Shao, site directed mutagenesis isn't really the right tool for this, because you're restricted by the size of the primers.
You could also save yourself some headaches by just getting your 200 bp product synthesized as a gBlock that rather than ordering a bunch of primers, I'm guessing getting a gBlock of that size would be roughly around the price as ordering several very large primers. If you decide to go that route you can design the ends of the gBlock to be suitable for whatever cloning method you end up using.
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