Bacterial two hybrid system - problems (Nov/11/2003 )
i just want to used the stratagene`s bacteriomatchII SYSTEM to study the map of protein-protein interaction of a bacterium,i should construct a radom peptide library.where can i find more detail material about the instruction for construction the peptide library?
Regarding the expression of your recombiant protein in pBT
The cloning can take a long time depending on the nature of your gene and the choices of the rescrition enzymes sites...
I inserted the sites by PCR and it works great. Moreover doing that way you get a lot of your insert. The yield of pBT is very low so you should use the low copy protocol for your prep
If the cloning is ok
1) you must check that your gene is correctly cloned in frame by sequencing
2) you should try to make a cinetic of induction with different concentration of IPTG and different times (various O.D)
It works great for me that way
You should also load a lot of your bacterial prep in order to see a clear band by western blot (I directly used an antibody against my protein, I did not used the anti lambda cI described by stratagen).
For the construction of your random peptide library you should read some protocols of phage display, I think that the molecular biology steps are pretty similar. One important point is the insertion of the sites for in frame cloning...
I hope it will help you 
Actually I have some problems with the yield of cDNA obtained with the "XR" kit and the recovery after the size fractionating
Any of you have advices 
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).
I am using BacterioMatch II two hybrid system to study my protein-protein interaction. is ur gene is toxic? check ur insert sequece to ensure it is inframe with the reading frame of the fusion protein. how do u clone ur insert?
the concentration of the His DO is u make a stock of 1g in 100 ml. this is considered 10X. add 50 ml of this 10X His DO into ur selective medium or nonselective medium, it will become 1X. I already consult the technical support from Stratagene. It works. I used it b4 and get the result.
good luck!
do western blot. the concentration is too low to be detected by SDS-PAGE stained with CBB. buy the lamda cl antibody frm Stratagene. actually, u can skip tht part for verifying the expression. go ahead to the cotransformation.