Who has experiance with the bacterial two hybrid system by stratagene?
We have severe problems with cloning and with DNA-preparation out of Minis and Maxis of the bait vector (pBT).

Who has experiance with the bacterial two hybrid system by stratagene?
We have severe problems with cloning and with DNA-preparation out of Minis and Maxis of the bait vector (pBT).

Hi. I'm about to do a library screening using bacteriomatch I and maybe bacteriomatch II. I've had no problems cloning my protein in the pBT. For mini preps i've used a qiagen kit with the apropriate modifications for low copy plasmids (10 ml culture for instance) and have eluted the DNA with only 20 microliters. If any of you has meanwhile completed your experiences with this kit can you please tell me any problems you've had and the alterations to the protocol you did to overcome them? Thanks.

Hi. I am using bacteriomatch II. Cotransformation with the control plasmids is easy and works. (but they don't grow if the "strep" is higher than 10µg/ml).
To purify pTRG-cDNAlibrary and pBT-Insert I use mini preps (qiagen) with the apropriate modifications for low copy plasmids (5 ml culture and elute the DNA with 30µl). Then, the number of cotransformat is really low. The big question is:
Does anyone know how much is the DNA concentration (µg/µl) that you get when you elute in 30µl from 5ml overnight culture?
Do you dilute the DNA the cotransformation?how many µl do you use?
I am very interested in contact with somebody using BM2HSII

Hi there,

which one works better, BacterioMatch or YTH? Appreciate your comments.

DZ

I have been using BacterioMatch I and II for the past 2 years. I have been very dissapointed with Stratagene as they clearly sold me a product which was just launched without the appropriate testing done on it. Thus the emergence of version II. I spent almost a year trying to clone two different baits in the pBT plasmid and although I managed it in the end it was extremely difficult. The bait and target plasmids are very difficult to work with. The only thing that worked for me in the end was using brand new plasmid from the company to digest and ligate. They are growing it in huuge cultures (20 L) at 30 degrees which obviously cannot be done in a normal lab. So I suggest that you do your initial cloning from the Stratagene original plasmid. After that it is easier to propagate. I would also suggest using 10 ml mini cultures instead of 5 ml for mini preps. You would then get around 0.5 ug /ml which is not very bad. Always dilute your bait palsimd and target plasmid before cotransformation to use 50 ng as the protocol suggests and follow the cotransformation protocol to the letter. The first version produced a huge number of false positives which I could only distinguish when a performed GST pull downs at great expense of money and time. Version II seems to be better but it begins to resemble more and more like a Y2H system. However it is quicker and the cotransformations are easier than the Y2H. Good Luck!!!!

I have only just begun to use the Stratagene Kit, and this is my first experience with two-hybrid systems. As for cloning into the pBT, I have not had any problems. With Qiagen mini-preps (using spins), I consistently get 50 ng/uL from 5 mL 24hr culture at 30C. I elute from the column in 50 uL.

For cloning, I have been digesting the plasmids with EcoRI and XhoI. I have been using PCR clean-up kits (Qiagen) between each step instead of phenol/chloroform extractions. With this small of a drop out, gel isolation is not necessary.

I have been cloning via PCR, using Phusion polymerase (high fidelity). I have engeneered sites into the primers, as one typically would. For ligation, I have been using the new rapid ligation kit from NEB without deviation from their protocol. I have cloned 10 genes in this way with success on the first attemp for each gene.

However, I have yet to recapitulate positive interactions published in yeast system. ...yet. I will continue evaluating the system.

Does anyone know of any other bacterial systems that are commercially available?

Zee

i want to know the concentration of His-dropout amino suppliment, the instruction manual did not tell that .

i have cloned my gene into the pBT-vector,but the problem is that i can not detected the express of the protein on the SDS-PAGE agar.the concentration of IPTG is 50uM.OD is 1.0.what is the problem?help me please.