problem in M9 media - m9 media (Oct/07/2010 )
hi every one,
I m trying to grow my e.coli strain in m9 media for overexpression of my protein.. i m using this media fr the first time.. when i prepare i m nt getting a homogeneous solution instead getting precipitation of small particles im media aftr avtoclaving.. the media is in brownish colour.. am i doing correctly .. does m9 media hav precipitates ??i stittred and heated well to get a clear solution bt aftr autoclaving there s some sedimentation..
THis is my media recipe
Expression of HIV protease A
Made 4 L of M9 Media:
27.2 g Na2HPO4
12 g KH2PO4
2g NaCl
4g NH4Cl
20 g Casamino Acids
pH 7.0
thanks in advance
the casamino acids may be your problem. either filter sterilize them then add them to the medium after autoclaving or filter sterilize the complete medium without autoclaving.
mmmm on Thu Oct 7 10:39:52 2010 said:
hi every one,
I m trying to grow my e.coli strain in m9 media for overexpression of my protein.. i m using this media fr the first time.. when i prepare i m nt getting a homogeneous solution instead getting precipitation of small particles im media aftr avtoclaving.. the media is in brownish colour.. am i doing correctly .. does m9 media hav precipitates ??i stittred and heated well to get a clear solution bt aftr autoclaving there s some sedimentation..
THis is my media recipe
Expression of HIV protease A
Made 4 L of M9 Media:
27.2 g Na2HPO4
12 g KH2PO4
2g NaCl
4g NH4Cl
20 g Casamino Acids
pH 7.0
thanks in advance
Hola, Probably the problems came from sterilize casaminoacids with salts. M9 formula is quite different (eLabProtocols.com). You have to autoclave separatelly the 4xfour salts ,MgSO4, glucose , casaminoacids.Before seed bacteria, mix the components , add sterile H2O .(if you donīt use glucose, bacteria has to use the carbon structure of aminoacid, and the pattern of bands could be dirtier , because you are using minimal media to have a more clean pattern, isnīt it?. Good luck
protolder on Thu Oct 7 14:03:58 2010 said:
mmmm on Thu Oct 7 10:39:52 2010 said:
hi every one,
I m trying to grow my e.coli strain in m9 media for overexpression of my protein.. i m using this media fr the first time.. when i prepare i m nt getting a homogeneous solution instead getting precipitation of small particles im media aftr avtoclaving.. the media is in brownish colour.. am i doing correctly .. does m9 media hav precipitates ??i stittred and heated well to get a clear solution bt aftr autoclaving there s some sedimentation..
THis is my media recipe
Expression of HIV protease A
Made 4 L of M9 Media:
27.2 g Na2HPO4
12 g KH2PO4
2g NaCl
4g NH4Cl
20 g Casamino Acids
pH 7.0
thanks in advance
Hola, Probably the problems came from sterilize casaminoacids with salts. M9 formula is quite different (eLabProtocols.com). You have to autoclave separatelly the 4xfour salts ,MgSO4, glucose , casaminoacids.Before seed bacteria, mix the components , add sterile H2O .(if you donīt use glucose, bacteria has to use the carbon structure of aminoacid, and the pattern of bands could be dirtier , because you are using minimal media to have a more clean pattern, isnīt it?. Good luck
Thank a lot.. i wil try as u suggested..
mdfenko on Thu Oct 7 13:29:09 2010 said:
the casamino acids may be your problem. either filter sterilize them then add them to the medium after autoclaving or filter sterilize the complete medium without autoclaving.
Thank u