14 replies to this topic

[Sandra]

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).

[alberto]

Sandra, on Nov 11 2003, 05:16 AM, said:

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'm going to start using this kit "bacterioMATCH II two hybrid system" by Stratagene. I'm quite unsure that it will be better than yeast one. How long are you using the B2H? Cloning into the pBT is your only problem till now?See you later.

[dinis]

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.

[alvhernan]

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

[dxz12]

Hi there,

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

DZ

[mardo]

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!!!!

[dxz12]

mardo, on Apr 14 2004, 01:39 AM, said:

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!!!!

Dear mardo,

Thank you very much for sharing your experience with me. I rather use Y2H now, does it sound right?
DZ

[zee99]

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

[yyzhang]

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

[yyzhang]

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.

[yyzhang]

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?

[dlesage]

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

[kooisk]

Sandra, on Nov 11 2003, 06:16 AM, said:

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).

<{POST_SNAPBACK}>

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?

[kooisk]

yyzhang, on Jun 20 2004, 07:27 PM, said:

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

<{POST_SNAPBACK}>

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!

[kooisk]

yyzhang, on Jun 28 2004, 07:18 AM, said:

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.

<{POST_SNAPBACK}>

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.