Site-directed mutagenesis failed - (Dec/10/2013 )

How similar are the primers to the target sequence?  Any bases that aren't supposed to anneal should be discounted from the annealing temp calculations.

There are insertions and substitutions in the primer. The annealed parts of the primers have 97,6 C Tm. The whole primers have 100 C Tm. Not too different...

Primers actually never get such high Tm.

The Tm is increasing with length, but only to certain point, then it stalls. Because such long sequence doesn't melt at single point anymore, but creates smaller melting domains. Software that calculates Tm doesn't take this usually into account, because it's not that predictable.

When you have long primers, they even aren't more specific. From a length around 25-30 bp the specificity doesn't increase anymore, because it's so long, that only 3' end of primers anneal (this is used when designing long primers with 5' noncomplementary sequence like a restriction site, you can just add the aditional bp to a normal primer and use the Ta as you would for the primer alone, the 5' end will just hang freely).

So, too long primers have no additional benefits (SDM primers have around 30, because they have an inside mismatch and both ends have to be long enough to compensate for the mispriming to be specific enough, but no more) but only more and more problems with secondary structure.

Also it's not commonly available to sythesize such long primers, or/and the prices are much higher.

Ok. Thanks for your comments !

If a quikchange mutagenesis reaction doesn't work the first time, I just go straight to overlap extension to make the mutation. I have found myself spending days trying to troubleshoot a quickchange reaction when I could have just done the overlap extension, which although a bit more involved than quickchange works beautifully and every colony will have your mutation. Plus you can do multiple mutations at once with overlap extension as long as they are far enough apart in sequence (>100bp....though if the mutations are less than that far apart you can just have multiple mutations in a single primer). When amplifying for overlap extension, I have had great success with touchdown PCR as well- nice clean single bands every time.  Just my two cents.  

OE is fine if you only need a short product with your mutation. But if you have an expression vector with a gene you want to mutate (ane ideally not mutate anything else, so the less PCRs the better) I can't exactly imagine how this would be possible with OE..

I did several "mutation standards" for detection of rare mutations with OE-PCR, it's fun, but I can't see how it could be usefull for a plasmid that you need to tranform.

I'm a little confused at what you are saying Trof.

I use OE all the time to mutate a gene (as small as 200bp and up to as big as 3.5kbp) in many expression vectors, whether its making short insertions, deletions, single codon changes, multiple codon changes, gene fusions....just about anything you want. Works better than any other mutagenesis protocol I've tried. It is a little more work of course, but 100% of your clones will have the mutation (assuming primers are made correctly) compared to the tradition quickchange which in my hands yields only about 70% of clones with the desired sequence, and that's only if the quickchange amplification itself works (it can be very finicky about having perfect primers...mutagenic primer pairs that will not work with quickchange usually still work beautifully in OE). And I would argue OE propagates fewer mutations than quikchange since you aren't amplifying the entire plasmid, only the gene of interest, which then gets ligated back into the original parent vector (or another vector of choice...another advantage of OE) prior to transformation. 

I see, so you actually mean doing OE-PCR only on the gene of interest, because OE-PCR gives a linear product and THEN ligating it into vector again. Well that is limited in several ways by the length of the gene and the ligation step itself prior any tests. That can be more tricky than Quickchange itself.

By the way, I did Quickchange only like three times and picking out dozen of random colonies I always got 100% mutated minipreps. Unless the digestion or PCR itself fails (like you were suggesting) there is no reason why there should be higher percentage of unmutated vectors.

And if you got good results with the same primers for OE-PCR but not for Quickchange, the main reason seems to be the secondary structure of your vector, which prevents amplification of the whole plasmid over the difficult regions. For that reason some types of vectors and not used for mutagenesis but instead the Quickchange is done in other plasmid and then ligated. Which is the similar approach as you use, but you can verify the sequence of GOI before you ligate to a different vector.

But still you maybe amplifing only small portion of plasmid, but with much more cycles. Mutation rate of the same portion of DNA (i.e. the gene of interest) only depends on the number of cycles, that may be up to 2x lower in Quickchange. The simple vectors, where mutagenesis is done usually don't have much more than GOI and resistance gene. And negatively affecting mutations in the resistence gene are selected out. For the complex ones, they are rather cut and ligated as mentioned above.

Trof on Wed May 14 16:21:16 2014 said:

I see, so you actually mean doing OE-PCR only on the gene of interest, because OE-PCR gives a linear product and THEN ligating it into vector again. Well that is limited in several ways by the length of the gene and the ligation step itself prior any tests. That can be more tricky than Quickchange itself.

I typically work with expression vectors, which are relatively simple but do contain a few more important bits that cannot suffer mutations by the polymerase that would not be selected by antibiotic resistance (lac repressor, rbs's, promoter sites, etc). Only in a single case have I ever had a plasmid coming from quickchange not express due to a change in one of these sites, so in general I agree the mutation rate of quickchange is low enough (or silent enough) that it shouldn't cause too much issue. In general though I'm not too worried about this working with high fidelity polyermases.

Bottom line for me: I try quikchange the first time...if I get a lot of colonies, great...I'll sequence 3-4 of them and can be pretty sure I've got what I want. If quickchange fails on my first try, I go straight to OE-PCR. No need to fuss around optimizing the quickchange which may or may not work, or wait a week to get new primers, or use different subcloning vectors and move mutants around from there, etc.

labtastic:

I actually may be forced to try this strategy :) Because now I have a vector I can't QuickChange and I can't switch the portion of the gene with the mutation by single cutters (actually there are only two available sites 3' of the mutation and one cuts blunt twice, effectively cutting out the complete Ori+resistance gene part and the second is copletely blocked by Dam and I can't use Dam^- strain), also vector doesn't have MCS (at least not now).

So I though of amplifying the portion of the vector including my gene from one single cutting site to the next one, which lies out of the ORF (that's why I can't switch the parts, I have my gene mutated in different vector, the second site is not there) incorporating the mutation in the process, and then cut it and ligate into vector.

With a sheer luck, the 5' restriction site is so close to the mutation, that is actually present in my QuickChange primer I used, with the required additional bp for successful restriction. So in fact I would need only one PCR to have a full product, and it's relatively short too, for validation.

Will let you know how it went :)