Hello,
I would like to ask how to grow E.coli which resists 50 or 100ug/ml of Blasticidin.
I have tried many methods by using low salt Luria-Bertain(LB) agar plate.
However, no colonies grow.
Let me explain what I have tried.
1) I used LB agar powder with Blasticidin, which made from Invivogen.
2) I dissolved the powder in a microwave for 2min 30sec because the powder cannot be autoclaved.
3) I poured the melted medium in sterile plates, and stored plates overnight in a dark 4°C(39.2°F) refrigerator.
4) You can see the information of the LB agar powder from this site( https://www.invivogen.com/sites/default/files/invivogen/resources/documents/flyer-selective-antibiotics-invivogen.pdf ).
1) Autoclave Low salt LB agar(0.5g NaCl, 1g Tryptone powder, 0.5g Yeast extract, 1.5g Agar powder, and 100mL of distilled water.
2) Before using the Blasticidin S HCl(10mg/mL) which freezes at -4°C(24.8°F), thaw it at room temperature.
3) Allow the medium to cool to at least 55°C( 131°F ) before adding the blasticidin to 100ug/ml final concentration. I used the Blasticidin S HCl(10mg/mL), which made from ThermoFisher.
4) Store plates overnight at 4°C(39.2°F) in the dark.
5) You can see the information of the Blasticidin S HCl(10mg/mL) from this site( https://www.thermofisher.com/order/catalog/product/A1113903#/A1113903 )
1) Autoclave Low salt LB agar(0.5g NaCl, 1g Tryptone powder, 0.5g Yeast extract, 1.5g Agar powder, and 100mL of distilled water).
2) Before using the Blasticidin S HCl(10mg/mL) which freezes at -4°C(24.8°F), thaw it at room temperature.
3) Allow the medium to cool to at least 55°C( 131°F ) before adding the blasticidin to 50ug/ml final concentration. I used the Blasticidin S HCl(10mg/mL).
4) Store plates overnight at 4°C(39.2°F) in the dark.
What should I do in order to facilitate selection of blasticidin resistant E.coli?
Should I make the final concentration of blasticidin at lower than 50ug/ml?
I have no idea.
Thank you for reading my problems, and I am looking forward to seeing your answer.
If you say that no colonies grow on your plates, the most likely explanation is that you are dealing with an E. coli strain that is sensitive to blasticidin (or, if you a trying to use it as a selection agent for cloning, that your cloning didn't work).
I would prepare your low salt LB in the autoclave and then add the Blasticidin when cooled to 55 degrees. I think it should be fine at either 50 or 100 ug/ml. Are you certain that your strain is actually resistant, or as Pierre Béguin suggested, if you are doing a cloning experiment maybe that is the problem, or the transformation. Do you have any controls to try?
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