Hi,

1)You can add more concentration of plasmid during Transformation

2) Check your Dh5 alfa cell with other  positive plasmid

3) If then also colony is not there use calcium chloride method of transformation

4) your idea is to transform plasmid of your interest so no need to transform chromosomal DNA Expression of gene of interest may require different expression vector.

Regards

Dhaval

Dear Silvia,

Hope this helps if I have understood you correctly:

Would firstly do a miniprep of the resistant Klebsiella and check how many plasmids exist. Then identify the plasmid which is carrying the resistance gene. Quantify the plasmid to a concentration best suited for transformation and then proceed to transformation in E.coli.

Cheers

Hi Silvia,

I can't help very much with the total DNA extract idea. But it is worth checking the following things:

1- If you suspect it is a large plasmid and low copy, try different competent cells more suitable for such plasmids.

2- Again if it is a large plasmid, and you are doing just minipreps, maybe it is not the best kit to extract such plasmids....just go old school to extract and precipitate your DNA plasmid.

3- You can run your plasmid extract on a gel, as Abhishek suggested, then work out how many plasmids are present, and rough size estimation...maybe you can do gel extract and use it for your transformation.

Good luck!

Thank you all of you for your answers and suggestions. I was wondering how I can check how many plasmids exist and how I can identify the plasmid which is carrying the resistance gene?

After the plasmid extraction I have just run a gel to visualize them and it is difficult for me to determine how many plasmids I have..I have different bands, but I can't distinguish if they are bands of the same plasmids or they correspond to different plasmids.

Anyway an image of plasmids visualized after extraction  is in attachment. They correspond to different Klebsiella isolates. I can't distinguish if the different bands are different forms of the same plasmid or different plasmids.According to this, how can I determine how many plasmids exist and identify the plasmid which is carrying the resistance gene? Any suggestion would be really appreciated.