Dear Sandeep

it depends from the quality of you digested plasmid preparation.

did you performed double digestion?

did you perfom the dephosporilation step? https://international.neb.com/applications/cloning-and-synthetic-biology/dna-end-modification/dephosphorylation

did you purified fuor digested plasmid from gel to remove background due to the non digested plasmid?

generally if you do all those steps the background coming from non digested plasmid or plasmid re-ligated is very low and screen 4-8 colonies from each location is enough.

however you can check the quality of your digested plasmid by performing a blank ligase, ligating and trasforming only the digested plasmid wirh out the insert.

however in General i invite you to consider PIPE cloning as alternative more simple cloning method. you can find informations about it on my blog:

https://proteocool.blogspot.com/

good luck

Manuele

Hello, similar topic were discussed here.

https://www.google.com/amp/s/www.researchgate.net/post/Could_anyone_provide_some_reasons_why_after_a_ligation-transformation_experiment_I_get_colonies_on_ampicillin_resistant_plates_that_are_not_correct/amp

Good luck!

Hi there,

Without running transformation controls along, it's hard to tell. These controls are:

1) ligation mix containing the digested vector alone without ligase (this control wil tell you if there is uncut vector in your plasmid prep and to what extend it will impact the number of clones)

2) ligation mix containing vector alone and ligase (here transformants will result either from undigested vector or from self ligation event)

Ideally you get no clones on both controls which means that basically any clone obtained from the complete ligation reaction will be positive (then screening a couple of clones would e enough)

If your ligation has been succesful you are expecting more clones on the plate than for these controls. The very crucial thing is that no or a very few clones has to be observed from control 1).

If the number of clones is significantly higher for our complete ligation than for control 2) it means screening a few clones should be enough to get the right one.

If you get comparible number of clones between control 2) and your complete ligation reaction it doesn't mean there is no positive but to get them you will have to screen more clones (in that case I usually start with 10 to 20 clones).

And finally always include the suitable controls for your PCR screening (empty cells, clone from control 1) if any, clone from control 2) if any) to get the PCR pattern of negative clones for comparison.

Also, I would like to include my experience. I think it also depends on the competent cells quality. I prepared new competent cells and transfected the same ligated product using old and new comp cells. I observed 10 times difference in the growth of colonies as compared to the new comp. cells.