Hi Zeynep Selvitopi . Based on your control, the problem is not your competent cells. Might be the ligated DNA concentration is too low which reduces transformation efficiency.

I usually use 100uL CC + 20uL (the entire ligation product). Toxicity might occur but I am more interested to get at least few cells take up my plasmid rather than having a lot of them taking up the plasmid. Once you get positive transformant, you can propagate them at suitable plate for stock.

My heat shock protocol I am using is 42 degree celcius at 90s, then on ice 2 min. My recovery rpm is 220. And I usually concentrate the recovered cells (8k rpm, 5 minutes, discard to leave 100uL and resuspend pellet).

You might want to try these paramaters and see if it works for you. I am using JM109 strain, shouldn't have any much difference in term of handling with your dh5a.

Also, are you sure your amp-plate contains 100mg? It should be 100ug. 100mg is way too high which will cause your dh5a not able to survive even if they taken up the plasmid conferring ampR gene.

Hi Zeynep,

I am wondering why you are mentioning a ligated product? Do you perform any kind of cloning or do you cut something out of your vector and you have to religate it?

This would be very important as when your ligation is not working properly you will most likely get no colonies.

Here it would be good to know what you are doing exactly and if e.g. during cloning/ligation you expose your vector to UV light which can severly reduce transformation efficiency.

However, if you are not working with a ligated vector but are trying to transform simply the vector I would thing that most likely your DNA is degraded ( I don't think it is a problem withyour protocol our your cells as your positive conttrol with the pUC19 plasmid is working.)

Check your DNA integrity on a gel (if you haven't done so already) - using simply a photometer is not enough (if you do so), because this might give you good results regarding concentration and purity even if the DNA is not intact.

And just for the record - usually it is not ok to simply say that your ampiccilin plate contains 100 mg or 100 µg of ampicillin.

This is not a concentration information - which would be the correct way to do give this information!

Ususally you should have a end-concentration of 100 µg/mL or 100 mg/L in your medium (both would be correct, if you do the math). To get this end-concentration often a stock is used with a concentration of 100 mg/mL and than dilute by the factor 1000 - maybe your are doing this as well.

However, if you use LB-Medium (with Agar) with the correct concentration of 100 µg/mL (or 100 mg/L) and you pour 10 mL onto you plate your ampicillin plate would contain a total amount of 1000 µg (or 1 mg) of ampicillin - hope you can see the difference to the information you have given (or Alif).

If you give a total amount of ampicillin (or any other antibiotics) on your plate you also have to give the volume of your medium (e.g. 10 m)l so that the concentration can be calculated.

I hope you don't think I am picky , however I think this is really important as this can lead to misunderstandings which can really mess up your experiments.

KR Dörthe

Hi Dörthe Andrea Kesper

Thank you for your reply. I am trying to transform the ligation product.

As you said, there may be a problem with the ligation. I visualized the DNA control on the gel beforehand and continued.

I adjusted the ampicillin concentration to 100 μg based on the medium I prepared.

For example, for 250 ml of medium, I added 250 μl from a 100 mg amp stock and prepared a plate with 20 ml.

Hi Zeynep,

I would really like to help you trouble shooting your problem, however to do so I would need some further information.

Maybe you could show a picture of the gel? You could upload it here if you are willing to share this information.

Regarding your ligation it would be helpful to know what you have done exactly.

Which enzymes/restriction sites were used for digestion of the vector?

Do you try to integrated an insert into the vector?

How did you set up the ligation reaction? How many vector did you use, what ligase did you use? How long and at which temperature did you perform the ligation reaction?

Did you expose the vector and/or the potential insert to UV radation.

There are many variables when doing a ligation, however with more inforrmation we might get at least get an idea how to improve you results.

KR Dörthe