Site directed mutagenesis - his tag - insertional mutations (Aug/14/2005 )

Hi All,

I'm new in this forum and this is my first post. I'm trying to obatan a His-tag tag version of a protein and I thought of using the Quickchange site directed mutageneis kit for that. The reason was facility and I didnt want to purcahse a new His tag vector since I had the Quickchange kit with me.

The problem I have is with the desing of primer

Option 1 is

Forward primer - 5' TAC TAC TAC GTA ...................... (29-45) 3'

Reverse primer - 5' GTA GTA GTA .................................(29-45) 3'

so that three of the histidines are encoded by the sequence of each of the two primers. Of course the mutational clone will contain TAC TAC TAC sticky ends and the reverse strand might (GTA GTA GTA) anneal and possibly influence the integration of the nicked strands

Option 2 is

Forward Prime - 5' TAC TAC TAC TAC TAC TAC GTA ......................(29-45) 3'

Reverse primer - 5' .......................................................................(29-45) 3'

In this case the whole hexa histidine tag is coded by one of the primers but the primers are uneven.

What do you think I should do ????? This is the first time I've done insertional mutagenesis and i wonder which of the above (or other) would be the best option.

And advice would be appreciated.

Thank you.

Dili

Have a look at my NAR paper - it is very easy and pretty foolproof.

Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

Daniel

DNA sequencing analysis programs

nice work Daniel!

I´m going to read your paper more carefully but in the meanwhile I just want to ask you how long can the deletion be with your strategy? Can I delete 50-200bp like an intron?


To Dili:

I think you have to design primers that are 100% complementary to each other and with the mismatches or mutations inside the primers. With different sequences in primers you must expect for the E. coli to correct mismatches after transformation and this could be randomly. I would try to use your original sequence and keep as much as you can. for his you have two codons and they are equally used in E. coli. the more mutations you need more length for the primers. you also can sequentially mutate with two sets of primers. so one set will have your 3 codons like in your fwd primer but in the middle, and the other set will have the other 3 codons but considering that a previous tri mutation occurred.

If I interpreted the paper correctly, you can delete as many bases as you like.

I would be interested in finding out what the maximum length you can use for tailing...

-Matt

Nori you can delete any number of bases you want - the only limitation is you must be able to PCR amplify the desired fragment (ie it wouldn't work too well on a 200kb BAC clone).

MisticMatt their is no upper length on the insert you can add as you can combine the SLIM approach with the SOE technique.

The primer designs you have listed look like trouble to me. The repeats look problematic in primer binding. Can you design a version with alternative HIS codons?

And, now that I notice, aren't the two codons for HIS CAT and CAC? I don't see either of those on your primers.

You have to use complementary bases for the tails or it wouldn't work.

There are plenty of PCR techniques that use self-annealing primer sets, including quikchange. You just use an excess of primers (that's pretty much the case in any PCR reaction, anyway).

-Matt