We are trying to transform a HygR construct into MACH1, using Hyg at 100 ug/mL as selection. However we keep getting a bacterial lawn even in the non-transformed control, using two different antibiotic stocks (one freshly prepared). We will next try DH5 alpha (it has worked well in the past) but we wanted to find out, has anybody out there had the same problem?
Hello, the Mach1 cell is one of the fastest-growing chemically competent strains currently available but I think it has ampicillin resistance only.
For hygromycin resistance you need to find E. coli which transformed with the hyg gene, which encodes a protein that detoxifies hygromycin B.
Hello, the Mach1 cell is one of the fastest-growing chemically competent strains currently available but I think it has ampicillin resistance only.
For hygromycin resistance you need to find E. coli which transformed with the hyg gene, which encodes a protein that detoxifies hygromycin B.
Karen A. Darbinyan: Sorry, I think I did not explain myself very well. We are trying to transform a HygR plasmid into MACH1, and the thing is, instead of well-isolated colonies we are getting a lawn, even in control plates where non-transformed cells have been plated. We first thought the antibiotic was off, so prepared a fresh stock and repeated the transformation, to no avail. Since this has worked in the past with other strains, we were wondering if perhaps E. coli W derivatives (such as MACH1) exhibit hygromycin resistance on their own.
Still, no biggie. I will check a K12 strain to see what's up.
Yes, I see only title ), some strains of E.coli can have has resistance to hygromycin. To be sure check before transformation the grow test on plates with hygromycin content in media.
Just a follow-up. Yes, it looks as if MACH1 has some background level of HygR resistance. It forms lawn at Hyg concentrations up to 150 ug/mL (at which JM109, an E. coli K12 derivative, does not grow).
Dear Dr. Alejandro,
Your views are accurate. I have experienced similar difficulties when I had used MACH 1. I initially doubted my antibiotic's potency. Later, while using other E.coli strains and the same antibiotic formulation, I happened to realize that MACH1, naturally has resistance to Hygromycin.
Dear Navnit Kumar Ramamoorthy as already mentioned the several strains of E.coli including MACH 1 has some resistance against Hygromycin, if you need strain without resistance can check ability grow on media with Hygromycin content.
Good luck!
Dear dr. Alejandro Martin
Very useful contribution
Best wishes
Dear dr. Karen A. Darbinyan
Good contributions
Best Regards
Dear dr. Navnit Kumar Ramamoorthy
Very useful contributions
BEst regards
Dear dr. Alejandro Martin
Very good contributions
BEst wishes
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