Seems, your mutagenesis would be easier only with normal pfu polymerase and transformation into DH5a or any common E.coli strains(since the size of the vector is just 4kb); So I mean to say it should work without any super kit and super compcells :(

You will get positive clones I guess:)

Gd luck.

Sorry for my wrong smily ;)

are you sure that your primers are purified in PAGE?

however you can replace the NZY medium with the SOB medium.

good luck :)

Thanks guys. And ayyappan its alright :) i got it. And yeah ill give a shot with dh5a.

Maria, now as for my primers i did not page purify, but i think that could'nt have been a problem right? Given that i was able to see my pcr product, even though faint.

And with the medium even i felt normal lb or sob would be more than enough.

Andreas as for the controls can you be more clear when you say you add all possible transformation controls? Im not getting you there.

Fredj i tried transforming with 1ul, 3 and 6 ( though i did not quantify in a nanodrop), but none worked anyhow. And indeed it is the quickchange kit i am also using.

In my experience, in some cases the page purification made difference

Did you check the PCR on a gel after your DpnI digest? I would suggest looking at it afterwards in case you are seeing residual plasmid template and not PCR product. If you don't see a band after your DpnI, then you need to optimize the PCR. Are you transforming with chemical or electroporation? We have found electroporation to be more efficient, especially for mutagenesis reactions. You could also try ethanol precipitating your reaction before transformation and double check your antibiotic in the plates and template.

When you amplify the entire plasmid with a pair of primers which have mutated sequence, you do not need a kit. Just phosphorylate the end of PCR products, digest the template with Dnp-1, purify PCR products by agarose electrophoresis, and ligate it. If you are worried about point mutations in the vector sequence by PCR, transfer the insert into the same vector after check the sequence of the insert.

Hello,

this strategy is very robust and in my experience as long as you have enough PCR product (after a complete DpnI digest) you can transform any bacteria you have. We have our home made competent DH5alpha and I take 7-10 ul of a 50 ul PCR reaction. I use LB only and it almost always works. Please don't forget to control your transformation using an intact plasmid as a positive control. Good luck!

I have been using the same Stratagene kits for mutagenesis and I had gotten the colonies post transformation, but I had seen my senior not getting colonies, so one it may be a problem with the Comp. cells, either use the kit comp cells only coz it depends on the vector you have cloned in. if the copy no. of it in some bacteria is not high then probability of getting mutated colonies is less. Plus you need to check little on gel post digestion if you have not been getting colonies. It might shoe band in gel but transformation can be a problem. Add more of digested DNA and still if it doesn't help, then you got to redesign your primers. But please check DH5 alpha comp cells with few micro litres of beta-mercaptoethanol when you transform, it should work. Good luck.

Hey Kalpita,

What beta-ME does with DH5alpha? I am curious to know.

I have the same experience as most people who have commented - we routinely perform QuikChange Mutagenesis using the Stratagene protocol, and transforming as little as 5 µl into 50 µl of homemade DH5a. Also, many of my colleagues use non-PAGE primers, and have gotten the right mutants.

I always check the presence and amount of DNA after DpnI digestion before transformation. In some cases where PCR was not giving good quantities of mutagenized product, it helped to prolong the elongation time up to 2 min/kb plasmid.

I think the transformation controls Andreas Moor suggested are

a no plasmid = negative control and original non-mutagenized plasmid control = positive control.

Hi Ananda, well beta ME is said to increase transformation efficiency for the most part and I too have observed it atleast in DH5a strain, expecially if your comp cells are lil old and you want good tranformants in terms of no. of colonies, it does help. I am still searching for the reference how it happens. May be membrane accessibility, poration in an effective way.

Make sure your primers are purified to high purity (eg. PAGE) as per protocol. This is the major factor leading to successful SDM

I actually have god results with this protocol. I never checked my PCR products on a gel and would not pay to much attention to it. I think the protocol even recommends to carry on ,even you don't see a band. I think it is important to use a suitable polymerase (e.g. PfuTurbo from Agilent). If you use large constructs you should check the manual of the polymerase for adjustment of buffer, dNTPs or polymerase concentrations as well as elongation and denaturation temperature and time (see PfuTurbo manual). You should also do not more than 18 to 20 cycles, in order not to introduce any errors and use not to much template (100 to 200 ng plasmid/50 µl PCR). After DnpI treatment (1h or longer) you should not use to much of your 50 µl PCR mix for transformation (I use 5 to 7 µl). I always use self-made DH5alpha.

like Sebastian mentions, size matters, 5uL of digested DNA, appropriate pfu enzyme, atleast 40 mM dNTPs so that they donot get exhausted and good primers plus fresh DH5alpha comp cells should work.

Transformation efficiency is never a trivial question, it so happens that for one gene and a vector your transformation works but for other it might not, so membrane composition and DNA uptake ability always matters.

Hello Markus,

Thanks for the explanation.

Yes I loaded a 10ul of the 50ul PCR reaction in my gel.

And I too came across the method of using any proof reading polymerase such as Phusion for this purpose and apparently this seems to be a cost efficient method, that does not demand super competent cells or a special kit of sorts. But apparently I will have to redesign my primers I presume(given that they have to be back to back, and not exactly complement each other, which is exactly the opposite to the ones designed according to stratagene guidelines).

We do the primer design exactly as Markus suggests, with good results.

guys hi again!

can someone please tell me the pcr conditions that you follow to get the right product.

I am quite confused with the different pcr makeups suggested by different sources. this is mainly regarding the concentration of template, primers, and dNTPs. My template has a conc of 250ng/ul, i have a 25mM and 2 mM dNTP mix, and my primers are of a 25pM working concentration (32 nucleotides in length with a Tm of 78 according to the stratagene formula).

Thanks to all you guys!

I finally made it. Got my colonies!!!! Yay!!!!!!! :) :) .

this time i had new primers designed with 3 nucleotides from the 3' end removed ( not sure if this helped exactly), and also I got back to the left overs of the kit, plus I managed to borrow some xl1blues from another lab. got more than a 100 colonies. But of course still i have not finished my race. got to screen my colonies now and lets see! I hope I get it right till the end.

Thanks again,

Cheers!!!!!

@ Markus,

I followed your suggestions and made lots of SDM very efficiently. 

Now I got a plasmid that is much bigger close to 10 KB. Do you think using same trick and just increasing the extension time will work? A colleague of mine tried several times with complementary primers without any success.  Is there anything that I should consider? I would be using Q5 hot start  from NEB.