How old are your competent cells? What method are you using for the transformation protocol? What strain of E.coli are you using? What do you mean by low transformation frequency? i.e. how many colonies did you get when transforming?

Normally, when frozen stocks of E.coli are stored for very long time the transformation efficiency decreases. Make a new batch of competent cells and try again.

Some strains could be more difficult to transform efficiently, for example, the strain BL21 forms a viscous pellet and the transformation frequency is lower than DH5a. For BL21 I always mix the plasmid and the cells thoroughly but carefully.

Electrocompetent cells are more efficient than heat shock competent cells, give it a try.

Hi Tam,.

Xi provided you with excellent explanations and advices.

If you want somebody to help you, you should write more details in your transformaiton procedure.

The control plasmid to see the level of E. coli comptetency should be 0.1 ng of the high-copy pUC19, and the only question that I'd like to ask you is 'do you perform a step of phenotypic expression (a recovery step)'? i.e. 1 hour growth in LB or SOC medium at 37^C whithout antibiotics? If you use other than ampicillin antibiotic, you have to perform a phenotypic expression.

Best and success,

yevgeny

One successful colony is enough for your following experiments.

Agree with Yau, getting one colony is sufficient if that colony contains the plasmid with the correct insert. There was once when I did my transformation, it also yielded low number of colonies (less than 10) but it contains plasmid with the correct insert, so it was considered as a success. Remember, the important thing here is not the number of colony you get on the plate, it's whether you get any colony with the plasmid and correct insert.

However, few colonies from the control plasmid is not correct. I would advise to make fresh competent cells.Then, take two or three control plasmids, examine them by agarose electrophoresis, and verify their concentrations. Use them for control transformation. If it is Ok, proceed with ligation reaction.

Tam Nguyen There are a few things that I would recommend you to consider

1. Are the competent cellls used fresh?

2. What E. coli starins you used?

3. Are the reagents used for ligation experiment working properly? Did anybody else in your lab have similar issues?

4. Did you check that your water bath or heating plate that you used for ransformation functioning properly i.e. the temperature it shows is the correct temperature? Sometimes this simple instrumental thing can result in such consequences

5. Did you verify that you have correct plasmid and correct insert?

6. Always remember it reallly does not matter how many colonies you get, the most important thing is to get one colony with correct insert ? Did you verify by performing sequencing that your insert has corrected been placed in the plasmid?

Hope it helps.