Id the antibody domain toxic to the cells or they recombine in the genome. If that is the case then try to change the strain.

TG1 is most commonly used for library preparation so i dont think bacterial strain is the issue...

Do,t you use the fast ligase? This is done only in 10-30 minute. Anyway you can check whether you have the successful ligation by per. Just take the vector primers flanking the inner. If you have the desired product then you know that ligation is no problem. aAlso You can check the ligation by running on the gel. If you get the shift compared to vector alone then you are sure that the ligation is not the problem.

If that is established then try to focus why you do not get the colonies. As I have said earlier some proteins are toxic to the cell so try to use a tightly controlled promoter vector.

Then obviously the strain issue you have already ruled out.

Good luck!

Transformation efficiency of your electrocompetent cells is quite low. I dont think they are good for library construction, but  its hard to believe that this would result in zero colonies.

How much DNA are you using to electroporateWhen I make libraries I try to do small ligations first (using 50-100 ng vector and the corresponding amount of insert) with the digested vector/insert to test wheteher they are able to ligate and the optimal vector/insert ratio. Once I have successful ligations, I go to the large ligation/electroporation with several micrograms of DNA.  

Do you have a control phagemid (preferably same backbone, but different ScFv) to check your system?

Which protocol do you follow?

Are you using 2% glucose in solid media (if your phagemid has the lac promoter)? This can minimize toxicity effects by repressing expression...

@wolfgang schechinger 

i have used control phagemid where human VH and VL was inserted and transformation was working fine. infact i got 10*7 colonies. i am following current protocols in immunology for library construction.

@Gertrudis Rojas

first i have tried transformation for 100ng vector and ~20ng vector for each electrophoration so it worked fine but when i did same at large scale then it never worked. My phagemid vector contains lac promoter but i never used 2% glu 2XYT-Amp plates . so could it be the reason for blank plates??

For reference / comparison, a protocol that is known to work: http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf

What do you mean by large scale, how much are you trying to electroporate? Which volume of cells, DNA amount and which kind of cuvette? How many kV? Which was your time constant in ms? Im trying to know whether there is something wrong with the electroporation step. Your cells are not good enogh for libraries (10-7 is a low efficiency), but should get at least some colonies.

Were the small electroporations done only with vector or with small scale ligation products? Perhaps your ligation is failing.

About glucose in the plates, I really dont think that this explains that you got no colonies at all. But with lac promoter phagemids, its a common practice to add 2% glucose to every plate and also to the liquid cultures, unless in the step of phage production. The goal is to keep repressed the expression of the foreign gene when its not necessary. This can make a difference when the product is very toxic.

@ Gertrudis Rojas 

Large scale means ligation of 6 ug vector with desired amt of insert( 100ng vector per 10ul reaction mixture), from this mixture i have used 5ul for elctroporation. two days back i did ligation for 100ng vector with insert and ran 2.5ul of this mixture on 1% agarose gel but i didnt get any shift of ligated band versus without ligation vector. still i did electorporation of ligation mixture( ligation mixture was diluted 3 times)  with 50ul of electrocompetent cells. final concentration of ligation mixture used per vial was 16 ng. i have used 1.8kV for electroporation. This time again i didnt get any colony even on 2%glu 2XYT-amp plates. 

So it seems that, besides having low-quality electrocompetent cells, you have a problem with ligation. I would suggest to optimize the small scale ligation by trying different vector/insert ratios and including always a control reaction with everything except the insert. This control should give you very few colonies if any.

For instance I normally use 1/8 vector/insert molar ratio when Im trying to ligate digested PCR products, but if I want to optimize, I try lower and higher ratios as well. If you have a successful reaction, you can try to reproduce it at a larger scale with the same proportions of the same digested vector and insert. If your digested vector and insert do not ligate at any ratio,probably  you would need to obtain better digestions. 

About electroporations, just be careful with the volume of reaction that you use to electroporate. If the products are purified or desalted you can use large volumes, but if you are working with the reactions without cleaning, only small volumes can be used without damaging the cells (a few microliters). You can check that by looking at the time constant after the pulse. If the time constant goes down to about  4ms or below, you have too much salt in the electroporation and this affects the cells and the electroporation efficiency.

@Gertrudis Rojas 

i have cross checked all parameters again. Upto ligation everything was fine then i have observed according to your suggestions that Time constant in terms of ms is below 4. so i think here is the main problem of getting minimum colonies. I ll pass the ligation mixture through column then do transformation because till now i was trying diluted ligation mixture.

Hi,

Try using 1 mm cuvette and 2.0kV. It works for larger constructs. Time constant should be above 4 but I have had many succesful transformations with tau close to 3.

Best regards,

Wes