Follow this protocol and you should be out of trouble..

http://mcmanuslab.ucsf.edu/protocol/site-directed-mutagenesis-stratagene-protocol

The 5' end of your primer is too short: T°m is only 38°C.

T°m of your 3' end is 53.8°C.

Usually the annealing step is performed at 60°C but I guess it will not work in your case.

You can try:

98°C 30 sec

18 cycles:

98°C - 20 sec

55°C - 50 sec

72°C - 30 sec/kbp

72°C 5 min

4°C hold

>I also like to note that I use normal LB during the transformation procedure.

No problem.

>is the buffer essential when using phusion polymerase and its HF buffer

No.

If you use stratagene Quick change kit or atleast the primers designed through the tool , it really picks up good primers. Like it is mentioned earlier, your annealing needs to be in the higher range. Usually we pick up the ones from 65-80 degrees.

Typically a flank of around 15-20 bases with the mutated nuleotide in the middle. size around 30-35 bp, GC % around 35-40 and Tm 70-80.

5 μl of 10× reaction buffer

2 μl (10 ng) of pWhitescript 4.5-kb control plasmid (5 ng/μl)

1.25 μl (125 ng) of oligonucleotide control primer #1

[34-mer (100 ng/μl)]

1.25 μl (125 ng) of oligonucleotide control primer #2

[34-mer (100 ng/μl)]

1 μl of dNTP mix (40mM dNTP) NB: if not from kit, you can use normal dNTPs used for usual PCR but take approapriate volume to make it 40mM like say we use 8mM in PCR and for SDM, we use 5uL of that 8mM dNTP to make it 40mM. It works well.

38.5 μl ddH2O (to bring the final reaction volume to 50 μl)

Then add

1 μl of PfuUltra HF DNA polymerase (2.5 U/μl)

Cycling Parameters for the QuikChange II Site-Directed

Mutagenesis Method

Segment Cycles Temperature Time

1 /// 95°C 30 seconds/

2/// 95°C 30 seconds these steps go for 12-18 cycles.

55°C 1 minute

68°C 1 minute/kb of plasmid length*

then HOLD

* For example, a 5-kb plasmid requires 5 minutes at 68°C per cycle.

Point mutations 12

Single amino acid changes 16

Multiple amino acid deletions or insertions 18 cycles recommended.

good luck.

Hello Kannan,

I would like to know if you are getting a faint band after the PCR reaction and if you are getting some colonies after transformation (but no mutants)?

Are you following the phusion mutagenesis kit protocol or the stratagene one?

Regarding the primer, I would look for having the 3' end free with at least 10 bases for good annealing, with that interaction it should work. But I agree with Kalpita that you need at least 15 bases to have a better annealing. Did you check hairpin formation of the primer?

The stratagene protocol it's pretty good. If you follow it, you shouldn't have major problems. But since you are using phusion polymerase, you will probably have to adjust the protocol a bit. I have to say that my best results were with Pfu polymerase.

I wonder if you can amplify at all your product, maybe you can do a standard PCR to see if the primers are working (35 cycles) or if the polymerase is in good shape.

Also, generally you add your DpnI enzyme directly to you PCR reaction tube after it's finished, you don't need to clean the product or change buffer conditions.

Although you have better recovery with SOC media, LB does the job pretty well. It worked for me.

Good luck

Hi Cecilia!

I once got a faint band but the transformation did not work (no colonies at all ). In all my subsequent attempts I have failed even to get my band ( of course i did not follow the same conditions as my first try )

I have been trying to stick on to the stratagene protocol, but using phusion polymerase.

As for the primers I think i need to re check their efficiency.

Hi Kannan,

I have successfully done hundreds of site directed mutagenesis. The protocol I use is adopted from the one described in the Invitrogen manual.

Design of the primer:

There should be 5-6 codons up- and downstream of your desired (point-) mutation and the primer should start and end with C or G. Important: check your primer for any possible secondary structures (e.g. here: http://www.premierbiosoft.com/netprimer/index.html), whereas a deltaG for hairpins below -4 kcal/mol is critical. In such a case just add another silent mutation in the primer to get a better value.

Polymerase:

I obtained best results with cloned Pfu DNA polymerase from Stratagene. The supplied reaction buffer is suitable to do DpnI digestion right after PCR.

Protocol (for one reaction, 50µl):

5µl 10x Pfu buffer

125 ng fw Primer

125 ng rev. Primer

50 ng template

2.5 U native Pfu

ad water to 50µl

mix well and split the batch into two batches (i.e. 2x25µl).

Then use a cycler with a temp. gradient and program it in a way that you have a 55°C slot and a 57.5°C slot (annealing temp.). Place the split batch in both slots.

1) 95°C 1min

2) 95°C 1min

3) 55°C / 57.5°C 1min

4) 68°C x min (depending on the length of the vector/construct to be elongated; for Pfu estimate conservatively 500bp/min)

go back to 2 (18x)

5) 72°C 10 min

Re-unify the two batches and ad 1 or 2 µl of DpnI (NEB, at 20 U/µl). Incubate 2h at 37°C w/o shaking!

Transform.

Done.

Happy mutagenesis!

Kannan explained that he wants to use Phusion polymerase ...

As I said, I did hundreds of quick changes successfully with this protocol, so why not to share it? There is no obvious reason desperately using Phusion instead of Pfu so go ahead Kannan!

Chris actually i did try the protocol exactly the way you have mentioned with Pfu and tried my best with the Stratagene protocol, and this did not yield me good results. That is why I posted my query here in the first place. I do appreciate your answer but this does not help me much. Since I began running out of the kit components, I wanted to do the experiments with the available ingredients ( primary alteration being that with Phusion polymerase) and after several attempts Im still not making it!

Hi Kannan,

we don't use a kit as well and there is no need to use a kit at all since quick change works fine just with the protocol I posted. If your quick change does not work at all I wouldn't consider the polymerase to be the reason for it (if it is not older than half a year), since nearly all proof-reading polys do work well (at least I tested four different ones from two companies, no difference in accuracy of the results). Your primer sequence looks good to me, no critical hairpins were detected. If you have no positive clones you should add more DpnI right after PCR or check whether the enzyme works out in the buffer you use for PCR (some suppliers use a poly buffer not applicable for DpnI digestion, in this case you first have to purify your PCR product prior DpnI digestion). As I already said, the Pfu reaction buffer from Agilent/Stratagene can be used right after PCR for DpnI digestion, no need to add another buffer, just add 1-2 µl DpnI in the PCR batch. Have you checked whether you have a PCR product after amplification (agarose gel)? Does it have the correct size (5 kb)? If so, it's most likely your DpnI digestion does not work out and you by chance picked only the clones transformed with your template not carrying the desired mutation.

Chris thank you again, and 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!!!!!