Your question is no clear. did't you get result in colony PCR? You may not get amplification in colony PCR if you take more sample( colony) or use high fidelity DNA polymerase for PCR. I can suggest you more if you describe your problem in detail with detail steps.

If you are using ampicillin and and your BL21 cells without transformation do not give rise to colonies you are just facing a problem with your PCR. You should be aware that you are not working with a high copy number plasmid. In general, if you transform a well-characterized plasmid, it is not necessary to check the identity after re-transformation.

To be on the safe side, I would perform a small DNA-prep. followed by a restriction enzyme digest.

Dear Seema

Dear Seemakgree that your problem is not so clear, because if you already extract and purify the plasmid from DH5alpha and you are sure that it is correct (by checking it with PCR screening and sequencing) it is strange that after trasformation you obtain empty colonies under antibiotic selection.

Did you found colonies with out insert, or with out plasmid?

Because If the colonies did not carry the plasmid, it probably due to problems with antibiotics selctions (age of the plates) becuase generally what may happen in case of toxic genes is basal expression that is toxic for bacterial growth and no colonies after trasfromation but not empty colonies.

If the colonies do not have inserts, it may be related to non homogeneity of your plasmid preparation. Did you perform the plasmid extraction form DH5alpha from a single colony?

ciao

Manuele

I want to clonned p65 in pet 32a vector ,so i perform pcr amplification ,digestion gel extraction and ligation . There i found colonies ,so i perform the colony pcr get the positive colonies.

So, for expression i isolated the plasmid that are positive in colony PCR and transform it into BL21(DE3) cells.

then i inoculate one colony for primary culture and do the protein expression but i found in SDS-PAGE only TAG is expressed.

Then again i transform that plasmid in BL21(DE3) cells ,this time before inoculation i did the colony PCR for conformation there i found ONLY one colony have the insert remaning dont have.

WHY is it so,pls explain.hope this time you understand the problem.

Don't care about the remaining clones and forget about colony PCR. Take the DNA from the positive clone -Transform again in DH5 - Inocculate around 6 colonies - Perform a mini prep - Save the DNA of a positive prep with the expected size of the insert - Keep this DNA for transformation in BL21 cells. No future colony PCR needed.

you have to send for sequencing after any cloning to ensure your insert is correctly inserted and to ensure its in frame ,after that you can try to express your protein ,just try to take it step by step 1)cloning okay go to second step which is the expression 2) check the expression if expression is okay then check the solubility then try to purify your protein but if you jump from cloning to expression without sequencing you won`t know if your cloning is correct or not as the PCR colony sometimes give false results ,i am usually using miniprep then mini digest tests then sequencing after cloning

Dear Mohamed Halawa I already done the mini digest and sequencing before expression and its seems good . Tell me what happen with my BL21 cells.

did you make mini prep ?? i am not understand why you did PCR colony before after the expression ,if you already sure that your insert in the vector ??anyway if the `Bl21 cells not working well you have to make the efficient test for your cells or make a fresh batch

Hello

Kindly look at the link below

Article Rapid assembly of multiple DNA fragments through direct tran...

Good Luck