Problems with Stratagene Site Directed Mutagenesis - (Apr/08/2009 )

I would check your NZY+ broth by growing an E. coli culture in it. I would check my plates by growing an amp resistant coli strain on my plates.

when I developed my molecule, had some difficulties in the visualization of the processed material. My problem is addressed through new strategies of primer size (decreases the amount ofbases). Try lowering to prevent looping of amino acids. good luck

Hello, I have recently ran into problems with SDM. I am introducing 2 mutations into a plasmid construct. The first of the two went smoothly. Here were my conditions.

50 µl PCR reaction

10X pfu reaction buffer 5 µl
10 mM dNTP mix 1.25 µl
Forward mutagenesis primer 2.0 µl
Reverse mutagenesis primer 2.0 µl
dsDNA template (150 ng) 2.0 µl
pfu Ultra fusion II polymerase 1.0 µl
H₂O 36.75 µl

TOTAL 50 µl

Thermocycler profile

95ᵒC – 4’

95ᵒC – 30 sec
50ᵒC – 1’ X 25
72ᵒC – 16’

72ᵒC – 7’
4ᵒC - ∞

The Tm of all primers is 62.8C. The GC content of the primers for the successful rxn was 47%.

Now, I used the same profile for the 2nd mutation and got no colonies. The Tm of the primers was 62.8C and the GC content was 57%. The template was the same and all conditions were the same. I think it has to be the annealing temperature. Would the 10% difference in GC content between the reactions cause the conditions not to work for reaction 2? Do you think it would help to raise the annealing temperature to say 55C?

Any input would be greatly appreciated.

Thanks

yean_ny_nie on Fri Apr 10 00:58:46 2009 said:

I use the KOD-Plus(my old friend used the TAKARA HS STAR Polymerase) instead of using the high price Strategene site-directed mutagenesis kit. I have gained about ~ 19 mutaed clones, and find it is so easy to make a point mutation. The key step is obvious the primer disigning and calculating the Tm. The Tm calculation used the method refferref in the manuscript in the Strategene kit. I ususally used the 18 cycles to amplify and usually 20-40ng per 50 ul reaction volume and 10 ul PCR products definitely make your to observe the clear band in ~1% AGE detection.

I want to upload a figure to you but I don't know how to paste it in this Bioforum.

From my accumulating experience, I feel the mutagenesis is so easy to indertaking and need all of your reagent in good condition.

The primer: need calculating Tm,well designed and should be synthetised at least with a minimum purity of PAGE purification grade, the dilution procedure is 100uM stocking solution diluted with TE buffer and dilutied with ddH2O(Millipore) to 10 uM working solution.

PCR is used uniformly followed the enzyme you used and used 18 cycles definitly working very well. I make 3 AA(amino acids) deletion and mutated to 3 Ala, point mutation and double AA mutation. All are work well. What's more, I just pick up 3 clones for sequencing cloning, the averaging positive clones ration almost more than 60 %.

So, take more cautious to your primer and PCR. But it should be mentioned that the Dpn I treatment is still a critical step. So load less template as possible is good for your mutagenesis.

Anyhow, Good lucky. If you want to have see my result figure you may sending me a email and disscuss something about this.

Hello all,
I am also trying to mutate a vector. My problem is that I am getting colonies in second day and they are growing very slow in liquid medium. I am wondering is it worth going to isolate DNA and go for sequencing ? i did not use any kit but used biorad iproof HF polymerase and transformed E Coli after DpnI digestion, purification and ligation. If anyone have any suggestions that would be a great help. Thanks.

There are many factors maybe accout for the clonies grow slowly in liquid medium: the medium is newly prepared or stored for a long time; and the host becterium physiological conditions. Moreover, which E.coli you used in your trials? DH5a usually grow slowly in my experience.
Of course, you should send the clones for sequencing to determine whether your clones is mutant or not.

Good luck!

fighter on Thu May 24 04:40:37 2012 said:

yes I was using DH5a. But this is something to do with the mutated construct because if I transform original vector, I am getting enough colonies by overnight. Transformation after mutation gives colonies after 48 hours. Any way I will isolate DNA and check. Thanks for your reply.