7 replies to this topic

[LacquerHead]

Hi all,

I need to add restriction sites to where a single BamHI site sits on my backbone to be able to ligate an insert into that spot, no MCS on this plasmid. I have not done this and am wondering if anyone has experience modifying the recipient vector with additional sites? Any rules/strategies for primer design to amplify my 8kb vector by PCR and add two unique sites? Thanks.

[phage434]

It's as easy as any other pcr. Don't forget to add 4-6 bp of junk DNA 5' of the restriction site, to allow the enzyme to cut. Make sure you purify your pcr product before cutting with the enzymes.

[LacquerHead]

The only complication is that the reverse primer anneals to a loxP site and its one of two loxP sites in the vector, so the primer is non-specific.

[phage434]

That is usually fatal. PCR will favor the shorter fragment. If you can extend the primer to the first base that mismatches in the two loxP sites, that is often sufficient. A single 3' base primer mismatch usually inhibits amplification.

[LacquerHead]

I would do that, and have designed such primers however the reverse primer would have to be about 33bp for to get to the first mismatch. Think this is ok? I can match up the forward primer in Tm appropriately.

[phage434]

This should not be a problem. After the first cycle, longer primers perfectly match in any pcr which adds a 5' extension. You're just doing it a cycle earlier.

[LacquerHead]

Thanks for the advice! I will try that. Seems easier to create new sites on my vector backbone that deleting a restriction site in the middle of my insert that I need for ligation.

[phage434]

Yes, and there's lower background from uncut circular plasmid transforming.