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Selecting bacteria with Puromycin

Cloning puromycin bacteria molecular biology

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#1 kwesi

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Posted 20 December 2015 - 10:58 PM

I want to use a plasmid with puromycin to select both bacteria and mammalian cells.

I learnt puromycin is not very effective for selecting bacteria. 

However, they say, under particular applications it can be used for selecting bacteria.

They mentioned the concentration as 125 ug/ml.

They say precise pH adjustment is required.

But they did not say the value of the pH.

Has anybody used puromycin for selecting bacteria? 

At what pH is the the drug effective against bacteria (e coli)?

 

Your response(s) will be very much appreciated

 

Sincerely,

Kwesi



#2 kwesi

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Posted 27 January 2016 - 11:33 PM

I had to do this by my self.

 

Interesting findings:

1. At the right pH, bacteria can be selected with 25-100ug/ml

2. At pH 6 and 6.5, the antibiotic was not active and the bacteria grew in 100ug/ul (liquid culture).

3. At pH 7, 7.5 and 8, the antibiotic was active (in liquid culture).

4. I chose pH 7.5 (50mM K-Phosphate buffer system). 

5. I made Lennox LB (5g/L instead of 10g/L NaCl)-agar. I learnt low salt is important for selection and supplemented it with 50mM K-PO4 buffer (1M, 20x stock). I autoclaved LB-agar and buffer separately, after cooling to 56oC, I added buffer and puromycin and then cast the agar into plates.

6. In this system 25ug/ml  puromycin worked perfectly.

 

I learnt auclaving LB and phosphate buffer may cause precipitations.

May be, tris-HCl, pH 7.5 can be used. In this case, it is possible to autoclave LB-agar and buffer in the same bottle which may be more convenient.

I will try this next time. I did not try what happens with non-buffered LB. Will try sometime.

 

I understand many people do not have the situation to use puromycin for bacteria selection.

 

Thank you all for willing to help.







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