Help with my PI - Asking to change to a different method. (Aug/16/2006 )

Hello,
I'm posting this in here because it sounds like the audience whose opinions I need. If it does not belong here my apologies to the moderators for thier needing to move it.

I am to express a gene for the purpose of making an antibody. My PI did this as part of her Ph.D work. When it came time to make an antibody for a different gene the task was given to me. She looked for the vector she used from her days of doing this. At the time this company had not paid its sales tax in TX so we could not get it. Instead she chose this other vector, from a different company (Invitrogen's pET 151-D TOPO). It is supposed to be the greatest thing since scliced bread! Topoisomerase carries out strand invasion cloning by cutting after a conserved sequence and ligating the gene of interest in directionally based on an overhang engineered into your PCR product. I have monkied with this plasmid for over A YEAR!!!!! I'm even at the point of quiting the lab because I have not gotten anywhere with this. I would really rather try to old school cut and ligate method. Does any one have any suggestions on how to ask my teacher to scrap this method and pay hundreds of dollars for a different one?

Any help would be appreciated.

Thank you,
Neil

I've used the directional Topo plasmids but with pcDNA3.1. It worked fine for me and was quicker than having using restriction enzymes sites etc. That said if your having problems and if it hasn't worked after a year I dwould efinitely switch to another cloning strategy. Cloning with specific restriction sites should take too long.

One of the problems with the directional TOPO is any sequence similarity in your PCR product internally or at the 3' with the sequence you have to put into your 5' primer before the gene specific portion (I think it's CCAAT) will mess up the TOPOs action.

If it's not worked by now if probably isn't going to work unless you change something. There are straight T-A TOPO cloning kits the disadvantage being only 50% of the clones are in the right orientation.

Every time I wanted to change my PI's mind, i made it really easy for him. I prepared a ~1-page document covering what the problem was, as well as 2 or 3 solutions. I would select the other option(s) so they were not really feasible.

Like most academics, my boss was always very busy. If I went to him and tried to talk through the issue, he was often distracted by other things. I found that if my boss found the 10 minutes to go through my document in private, he almost always went for my option. So easy to manipulate, and I didn't even have to use my Jedi powers.

Every time I wanted to change my PI's mind, i made it really easy for him. I prepared a ~1-page document covering what the problem was, as well as 2 or 3 solutions. I would select the other option(s) so they were not really feasible.

Like most academics, my boss was always very busy. If I went to him and tried to talk through the issue, he was often distracted by other things. I found that if my boss found the 10 minutes to go through my document in private, he almost always went for my option. So easy to manipulate, and I didn't even have to use my Jedi powers.

I always explain to my PI abt the problems and also tell them the amount of time and money which could b wasted. I tell my PI of an alternate and if he thinks its too expensive, I have to wait but will get t eventually. But if my experiments r going to stop bcoz of this stuff, I usually get it.

i think first point about relationship between student and PI (and also boss/employee) is the ability of discussion. In your case, discussing about your concern and the fact you want to try also standard ligation cloning process won't cause a crash. According to swanny, you can bring him small written sum up about the situation, and the solutions you're thinking of.
Go ahead for it.