Berglund:alternative method

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So, the first method allows you to actually insert in a different intron, different exons, etc without issue. This alternative method would allow you to make mutations, just not such large wholesale mutations.

Here, you would be working with the same DEK exon, intron, exon combo.

Let's say that your interest lies in the 3'splice site area (right before the second exon). This is what you would do.

You would design a normal short 5' primer to act as your forward, instead of a large one.

Then you would use Primer 4 as your first reverse.

Your template would be genomic DNA (in this case from human).

Do about 10 rounds of PCR off of 100ng of genomic DNA in a 30µl reaction (more rounds because this is now real life, not an artificial construction).

Clean up the PCR by gel purifying the product (need to get rid of the genomic)

Now, take your short forward primer and do 10 more cycles with primer 5 (30µl reaction)

Now, increase the volume of your PCR reaction to 100µl, adding more short forward primer, plus adding primer 6 (this dilutes primer 5 and allows primer 6 to do it's job).

Why I designed it this way:

Primer 4 is the primer of interest. It spans the 3'end of the intron. By using this primer, you can also use other primers(4, 5, or 6) that are mutants, just hook 4 with 5 and 6 and you are good to go. By doing it this way, instead of short forward primer with primer 6 on the genomic, you are also testing to see if you can get this to amplify up right using these primers. There are lots of options. Imagination is all you need.