Hello Tahera,

From what you say, it seems that you master the colony PCR technique, thus the problem is that your cloning didn't work and what you got are most likely colonies with empty vector. This could be due to several reasons, like poor digestion of the vector, poor quality or insufficient quantity of the fragment to be inserted, inactivation of ligase etc. Two tricks may help to reduce the background of colonies with empty vector:

1) Most vectors contain a multiple cloning site (MCS). If possible, choose to clone your insert between restriction sites that lie at either end of the MCS and design the amplification primers to include these same sites (asymmetrical cloning). If the vector undergoes complete double digestion, it will not self-ligate.

2) As 100% double digestion is difficult to achieve, the background can be further decreased by treating the ligation mixture before transformation with a restriction enzyme whose site was originally present in the MCS between the two restriction sites chosen for cloning.

Of course, the strategy should be designed avoiding any restriction sites that may lie within the sequence of the insert.

Good luck

Pierre Béguin

Hello,

Thank you for your suggestion and for helping me, but I tried to change every step and renewed again with no result. I will try one more according to your suggestion, hopefully, can get the result.

Thank you so much

Dear Tahera

which kind of cloning approach did you use?

Restriction enzymes and ligases? Which enzymes?

Did you purify the digested plasmid from agarose gel or do you simply perform a coloumn purification and de-fosforilation?

Did you perform a transformation test also with

- only digested vector?

- only PCR insert ? Did you amplified the PCR insert from genomic DNA or from a plasmid?

How big is your insert?

From your result it seems that your ligase do not work and the colonies that you have are associated to background from the not digested or re-ligated plasmid which is possible, if the fragment is very short, that do not show any amplification in the PCR (amplification may co-localize with primers)

Manuele

Manuele Martinelli

Hello,

I am doing cloning using a bacterial system, and gene transformation using PJET1.2/blunt plasmid vector.

and also using T4 ligase for ligation.

Thanks for your answer.

All of the above points are important, but also check the antibiotic because it is very important in the efficiency of the PCR colony.

Seyed Amir Hossein Mohammadzadeh Hosseini Moghri Thank you for your answer and suggestion.