Mutagenesis, deletion - what is the best kit/protocol (Jun/14/2011 )

Hi,

I'm trying to make a 350 bp deletion in my gene sequence, I'm wondering what is the best kit that I can use; I know that Stratagene sells several mutagenesis kits; they claim that even a deletion as big as this could be designed in the primers; but I'm sure it will be challenging if ever possible! the other kit which I'm using right know is Phusion (Finnzymes). the colonies that I have screened so far in addition to the desired deletion, also have several mutations just at the border of the deletions and not at the other parts of the sequence !!!! the primers do not contain those mutaions and I have absolutely no idea what can cause this !!! I'm really confused ! is there any other protocol or method that you may suggest?

Thanks

Are the colonies all with the same mutation? This may suggest that your source sequence is mutated.
I usually use the cheapest (homemade) Taq around, IMHO it doesn't really matter. It is more important that you have a good primer pair for mutagenesis.

Your primers *DO* have those mutations, although you didn't put them there. You might need to use hplc or gel purified oligos, or clone and sequence more examples. Oligos are very impure.

I think you are right phage434, I can not think of any thing else except primer impurity since in most of my sequenced clones there are similar mistakes in the primer site! But how could the PCR work with so many mismatches!? I guess since they are mostly in the 5' end! Now I'm gonna start over with HPLC purified primers, I didn't think it is necessary since primers were only 20-23 nucleotide long !!! and we never had such a problem before.

Hi Opal,
I dont know much about commercial plasmid but did you ever heard about using Heterozygous recombination?? i guess it will be good choice to delete 350bp, even can delete bigger size than yours. The plasmid that she used was PDM4, but i constructing my own plasmid, which also to delete my gene around 450-500bp. Deleting short fragment is easier to do than what my senior has done...it's about 1kb.

Evanescence on Thu Jun 16 15:25:06 2011 said:

ok i will give you the link..to download the journal, actually i am about doing the same but i designing my own plasmid for that purpose...if you want me to share the journal directly will share with you...in other words, if u need urgently, try to type "mutagenesis, sacB" it's key words...sacB normally used as counter selection for this mutagenesis...you can send me direct message if you want we will discuss later but now i am in the lab and need do some work..lol...if anything feel free to ask..we still in learning time...

Cheers

Evanescence