I prepare and use Stbl3 in the same way as DH5a without any problems. I would say that Stbl3 are even more competent. I think you should try to prepare a new chemically competent Stbl3. I use this protocol: http://www.princeton.edu/genomics/mcclean/protocols/Inoue-Method-for-Preparation-of-Competent-E-Coli.pdf

Hi Peter muller, I am using exact same protocol to prepare Stbl3 competent cells. I am getting very few colonies after transformation with 12.5 kb sized vector. When I ligated a 752 bp fragment into 12.5kb vector and transformed into same cells I am not at all getting positive colonies. All types of my ligation experiment with this 12.5 kb vector never gave any positive colonies in STBL3 cells.

There's no reason why a plasmid of that size wouldn't transform into Stbl3s.

Are you attempting to transform a ligation into the Stbl3 cells or are you just retransforming an existing plasmid. If it's the latter then you have a protocol problem because that should be incredibly efficient.

If it's a ligation then you probably have ligation issues unless your transformation works fine in DH5a and not at all in Stbl3. In which case I'd be blaming that particular batch of competent cells.

Thank you Jill Muhling, i will try it with dh5alpha , i did not try it yet because there was so much literature saying that it cause recombination to lentiviral vectors.

My issue was with ligation. Even when i transformed control vector (without any ligation or restriction) to stbl3, it  gave very few positive colonies. 

Ligation details: 

Restriction was done using Age1 (HF) and Xho1 in cutsmartbuffer. ----->Eluted the product from gel---->My insert size is 752 bp and vector size is 11500bp ,Ligation using T4 ligase (16 hours incubation at 16 degree celsius) --------> whole 20 microliter ligation reaction was treated with 100 microliter competent cells-----> kept on ice for 30 min-----> gave heat shock for 42 degree Celsius for 45 sec.--------->kept in ice for 5 min------> plated on Low salt lb plates.

OK this sounds like a ligation or a transformation issue - you CAN use DH5a cells for LV plasmids if all else fails. Just be sure to check carefully for recombination using restriction digestion and sequencing if possible. RecA+ E. coli dont always cause recombination in LV plasmids, it's just something you have to watch out for - I have a feeling that some plasmids are worse than others.

If you're hardly getting any colonies using the control vector then you have an issue - you should be getting a lawn of bacteria. Your competent cells may be bad (are they bought or home-made?) Also, did you allow your transformed bacteria to recover for one hour with no antibiotics at 37 degrees after heat-shock? 

yes, i did allow to recover for 1 hour. Since i doubted my competent stbl3 cells, i had once grown it in a plate with no antibiotics and i could get numerous colonies. But when i added control vector (12.5kb) i could get less than 10 colonies. later when i did ligation I got no colonies. This is my status.

Sometimes the bacteria with lentiviral constructs grow very slowly. Therefore, I grow some plasmids in half a dose of antibiotics, for example. Ampicillin, 50 ug / ml. Another possibility how to prevent recombination  is cultivation in DH5a at lower temperatures 25-30C. The important is that you must always check the plasmid integrity by restriction digest and run it on gel. 

Hi Peter Muller,

Thank you very much for your suggestion,  I will try using 50 microgram/ml of ampicillin for selecting stbl3 bacteria after ligation. I was always adding 100 micrograms/ml because that concentration was working fine with Dh5alpha, but I never thought of reducing the concentration of ampicillin until now. Thank you.

Do you recover with SOC?

Also , shake slowly when you are recovering....Usually I do 150 RPM or less .

How many colonies do you get for vector only and for vector plus insert?

How much DNA are you using for the ligation and how much do you add into the competent cells? When working with big fragments too much DNA is not good for transformation .

Hi Angel rivera thank you for your suggestion, While recovering I am shaking stbl3 cells at 200 rpm. I got colonies around 4-8 for control vector and no colonies for vector + insert.  I also used different ranges of concentration and I got colonies when I treated 1 ng dna (onwards). I am not getting colonies when I use vector at picogram levels . Therefore, as Peter Muller suggested I am trying to recover cells at lower ampicillin concentration.

We typically heat inactivate T4 ligation product before transformation and never use more than 1/10 of the volume. So if you had 100uL of competent cells, then use no more than 10uL ligation product. I hope that helps.

Hi Dany KO thank you, Yes I had inactivated the ligation and proportion (DNA: Competent cells ) was within that limit (1/10 th of the volume).

Hi ,

I tried inserting the cloned vector (apprx 12.5 kb) into stbl3 and plated them in 50 microgram/ml ampicillin plates. But i could not get any positive colonies.

Subsequently, I have also cloned the same vector into DH5alpha cells. I got colonies in ampicillin (50microgram/ml) containing plates but when I screened the colonies with restriction enzyme (double digest) , I did not find the insert inside the vector.  

stbl-3 is meant for repetitive DNA or Lenti-vector amplification, It is suggested to use high ampicillin concentration 100-200 ug/ml and 25-30 degree C or at room temp for 24-30 hrs incubation.

Hope this would help. 

Hi Rahul Raghavan .TV ,

I have exactly the same problem with you. Have you got positive colonies by now?

Thanks!

P.S

I have tested all the parameters and found the reason. Now I got more than 100 colonies. The key ingredient is SOC medium.

My protocol is as below:

- Ligation at 25 degree heater for 2hrs

- 35 mins incubation of stbl3 and ligation mix

- heat shcok at 42 degree for 45s

- IMPORTANT: Add 250uL SOC medium for recovering at 37 degree, 250 rpm for 1 hour.

- spread the cells

might need to grow at 30 degrees after plati to stop strange homologous recombination for happening

thanks