Genome walking services - what company to use? - (May/31/2008 )
I am considering spending some serious cash and using a genome walking service. I've seen a few advertised online but I am not sure who to go with.
Here are some of the companies advertising genome walking services:
3) Ocimum Biosolutions
I have never used such a service although I tried genome walking myself 2X unsuccessfully using the BIOS&T APAGOLD kit.
If anyone has had experience with any genome walking service (good or bad) could you please let me know?
I can imagine what a service does, but I can't imagine what is in a kit for this. Primer walking just sequences with one (or two) primers outward from some known region, and then re-synthesizes primers from the newly sequenced area(s). If you are fussy, you re-sequence in the reverse direction at the same time. You go about 600-700 bp per primer per reaction, so it takes a while, but it is very straightforward and I can't imagine what you need a kit for.
I guess that the kit makes the reverse primers (the ones outside your known sequence) more specific than just using short random oligos as your reverse primers. I am thinking that is important if you suspect that you have multiple copies of your gene in the genome. Otherwise, how do you know that you are walking the right one?
I my experience, the easiness of using a kit relies on the construction of a DNA library with different restriction enzymes that you can use for a lot of PCRs, ensuring repeatibility of your technique. Furthermore, you use your own designed primers (either forward or reverse) with the adaptors and if you think you gene is multicopy you will se bands in different libraries and after sequencing you will be able to know what is upstream and downstream from them. I have never used a service so i can't give an opinion about them.