can it be selective for YPD agar to have an antibiotic such as ampicillin in order to grow saccharomyces cerevisiae cells that may contain protein expression vector (pklac2) which has amp genes? Thank you.
The above answers are all correct, be sure to add the antibiotic after the medium has cooled somewhat from the autoclave.
I would however like to add that ampicillin does not function to select plasmids in yeast. It is solely used for selection in bacteria. While there are a few antibiotics used for selection in yeast, most often plasmid selection is done by the complementation of auxotrophic markers.
Hi,
You can try adding Amp. before pouring YPD agar. Shouldn't cause any problem since the yeast strain possesses Amp resistance genes. It wil only help better selection against contamination.
In some cases, a mixture of antibiotics can be useful. Please, also note that unlike other antibiotics, Chloramphenicol can be added before (!) autoclaving making platings more convenient in many cases.
Of course, but you must be careful with the temperature of medium. If it is too hot, you are going to destroy the antibiotic
Cooling down to 55°C is enough before adding the antibiotics. Otherwise you might get problems with pouring. Use syringe filter of 0.2 µm attached to the end of a syringe.
Suggested protocol recommends cooling to 55-60dC prior to addition. For detailed medium make up and additions see: https://sites.google.com/site/sciencebridgetechsites/sop-list/rnai-sops/media/making-lb-agar-with-ampicillin
Hi
I think you can do it and it helps the antibiotic spread better in your medium. But I recommend doing this after cooling down to 45-50 degrees centigrade. This temperature is a point that you can hardly tolerate it when touching the flasks by your hand.
You can ensure the antibiotic solution is sterile (and yes, it needs to be) by using filter sterilization (0.22um filter is best) prior to addition to the sterile medium.
I prefer to add antibiotics when the agar medium is just comfortably warm and not solidified. You may perform a cheek test !!! Simply touch the flask to your cheek, if your are comfortable add the antibiotic, mix it well and proceed further.
You can do it but the best way is to pour when the agar is just warm ready to pour on the plates
You can add the antibiotic to the agar before it solidifies and care should be taken to see uniform mixing of the antibiotic.
The above answers are all correct, be sure to add the antibiotic after the medium has cooled somewhat from the autoclave.
I would however like to add that ampicillin does not function to select plasmids in yeast. It is solely used for selection in bacteria. While there are a few antibiotics used for selection in yeast, most often plasmid selection is done by the complementation of auxotrophic markers.
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