Hello, I do not know about Takara MutanBEST kit but generally SDM for a plasmid involve two primers complementary to each other and the mutation is kept in the middle with flanking 15-20 bp on both sides. The PCR is carried out as usual and after DpnI digestion transform DH5alpha with 2-5 ul without any further treatment. From your diagram it seems that the Takara MutanBEST kit uses primers which are not complementary to each other and you need to do blunting/ligation. My feeling is same as yours that the problem is in blunting and ligation. You have to repeat blunting / kination / ligation. The other option is to design a Reverse primer complementary to Forward primer that was used for creating mutation and PCR again. After PCR use DpnI restriction enzyme to eliminate the template plasmid followed by transformation of DH5 alpha. No need to buy SDM kit. Use any good proof reading polymerase such as Pfu/phusion/kod etc. I hope this will work if blunting / kination / ligation are the cause of the problem. Good luck

I use Quik Change™ Site-Directed Mutagenesis Kit, it works fine, just PCR, digest with Dpn1, then Ecoli transformation.

Hello,

I indeed considered the codon usage bias when choosing the mutated site. But I guess this was not so important in my case.

I'm not using any mutagenesis kit. Homemade protocol works perfectly! My protocol is roughly:

1/ PCR. Be careful to design at least one primer with a 5'P otherwise blunt mutation will not work

2/ purification with QIAquick PCR purification kit (Qiagen)

3/ Digestion of the PCR product with DpnI in order to get rid of the non-mutant template DNA

4/ Gel purification to select your band of interest

5/ Ligation with T4 DNA ligase

6/ Transformation of 4-5µL in self-made Mash1 bacteria.

Hope it will help.

It is better to use stratagene kit, it works really well and easy to follow. No need of separate kinase/ligation reactions.

看你是厦大的，我就直接用中文了。现在都用quickchange kit。我本科也是厦大的，在厦大做点突变的时候，我并没有用kit。通常就用普通的takara的高保真酶。

大概的protocol。

1. 设计引物，突变位点左右各15bp大概，所以设计30bp的引物就行，反向引物和正向引物完全互补OK的。

2、PCR

3、DpnI

4、直接做transformation。（因为引物这样设计已经会有很大的粘性末端了，不需要做任何连接就可以直接转了）。

ps. 只要有PCR出来band来一般都会成功。你给的图的protocol反而麻烦了~希望有帮助。

Hello, I do not know about Takara MutanBEST kit but generally SDM for a plasmid involve two primers complementary to each other and the mutation is kept in the middle with flanking 15-20 bp on both sides. The PCR is carried out as usual and after DpnI digestion transform DH5alpha with 2-5 ul without any further treatment. From your diagram it seems that the Takara MutanBEST kit uses primers which are not complementary to each other and you need to do blunting/ligation. My feeling is same as yours that the problem is in blunting and ligation. You have to repeat blunting / kination / ligation. The other option is to design a Reverse primer complementary to Forward primer that was used for creating mutation and PCR again. After PCR use DpnI restriction enzyme to eliminate the template plasmid followed by transformation of DH5 alpha. No need to buy SDM kit. Use any good proof reading polymerase such as Pfu/phusion/kod etc. I hope this will work if blunting / kination / ligation are the cause of the problem. Good luck

Hi,

From my experience, plasmids after SDM are relatively hard to transform into DH5alfa. you should try ultracompetent cells (I used NEB5alfa, and even then only got several colonies). The Quik Change kit works great, and it comes with ultracompetent cells, so you can defiantly use it to.

Ligation efficiency is always problematic. The simplest, non-kit procedure is to perform megaprimer mutagenesis followed by MEGAWHOP PCR. This requires only a mutagenic primer and one of your existing flanking primers. If you want to order two mutagenic primers, then MEGAWHOP alone (quik-change) will do the trick. Protocols for megaprimer and megawhop can be found on our wiki at capsicum.colgate.edu/chwiki. We transform into ultra competent JM109 cells (zymo research) and prepare archival plasmid from there. Then you can easily transform into any strain.

Considering codon usage is a very good idea to make sure that mutant protein will be expressed as close to wild-type levels as possible, although codon usage certainly will not affect the mutagenesis itself. I have always used the QuikChange method for mutagenesis (I generally buy separate components and use the method), which does not include ligation. I agree with Daniel that supercompetent/ultra competent cells are very important. I use XL1-blue, but there are many others such as JM109 that should work). The size of your plasmid also is important to consider- the larger the size, the lower the transformation frequency will be. Stratagene has a specific QuikChange kit for longer plasmids (XL). A complete lack of colonies can also indicate other problems, but if you saw a product after thermocycling, then most of these possible problems are less likely.

I agree that blunt ligation of big plasmid DNA is inefficient. I suggest you to increase your DNA, reduce ligation volume and do overnight ligation at low temperature. As suggested by others, transformation efficiency is low in this case, if possible, use ready-made high-efficiency competent cells.

Wish you good luck.

Hi, we use the protocol from http://www.ncbi.nlm.nih.gov/pubmed/15304544. This approach produces the whole vector with the desired mutation. We were successfull with vectors up to 10 kb. We then transform into E. coli TOP10 made competent by this protocol: http://openwetware.org/wiki/TOP10_chemically_competent_cells. You can also use this protocol to make DH5a competent. Usually we get an efficiency of 0.5-1 x 10^8 cfu/µg DNA, which is quite good. All this gets you to the desired mutant within 5 days. Good luck,

Magdalena

Lots of good suggestions to follow in the comments above. Many protocols work; Quikchange is highly efficient and has the benefit of fewer steps (no phosphorylation or ligation required). However, you need to make sure your template DNA was grown in a bacterial strain that is dam/dcm +/+ (do NOT use JM110 or SCS110, for example).

If you choose to pursue the blunt ligation route, take care not to use too high a DNA concentration in your ligation. This will cause vector-vector ligation instead of self-ligation, and you will get no colonies. Limit yourself to 10-20ng of vector per ligation reaction.

Finally, assess carefully whether you really need to remove PstI by SDM. Many people assume that if you want to use a restriction site for cloning, it cannot occur within your insert. However, there are simple work-arounds for this problem that do not require SDM. Options are partial digestion, or 3-piece ligation, where you effectively mask your internal PstI with another unique restriction site internal to your cDNA. Would be happy to provide protocols for these alternate approaches if you need them. Good luck!

I would use the Stratagene Quickchange mutagenesis kit as others have suggested. Its expensive but it does work most of the time.

As to whether you need to consider codon bias, that depends on whether you are planning to go to mammalian from bacterial expression or vice versa. You can buy bacterial cell lines that will correct for codon bias (e.g. Stratagene Codon Plus or Rosetta Gami).

Personally though I prefer to do overlapping PCR by generating a 5' and 3' parts of the insert and then using end primers containing cloning sites to generate a full length PCR product which can then be digested and cloned. Slower, but far more certain to give a correct result. Also it allows you to have a full length PCR product that you can clone into different vectors if needed simultaneously

Vik

Dear all, thanks a lot for your detailed sugestions! I really appreciate that.

Just as Qaiser has suggested, I plan to repeat the blunting/kination/ligation again. If it fail, I will consider to design two complementary primers, PCR again and digest with DpnI, just like all your suggestions. I think I'd better order these enzyme first.

As for Quik Change™ Site-Directed Mutagenesis Kit, it seems this kit is effective but expensive. Thus I will put it in my nonpriority list.

Daniel Zhitnitsky and Lucy: mentioned the importance of competence cell. I will also put this aspect into consideration.

Daniel Cohen: recommanded to avoid a too high concentration of DNA in ligation, which might be a cause to no colonies. You also kindly suggested to think of the necessity of SDM. Actually I am going to construct a BioBrick using this DNA. The four standarderized restriction sites, however, are EcoRI, XbaI, SpeI and PstI. So I thought I have to eliminate PstI in the target gene. However, your idea is interesting but I could not fully understand the concept. There might be others who also find your method attractive. Would you please give us more details about it?