Hi y'all,
I just wanted to get your feedback on my troubleshooting of this plasmid. Any suggestions would be great. Thank you in advance.
We can successfully transform DH5alpha cells with the pMX-puro plasmid and we see colonies on LB-Amp (50 ug/ml Amp) plates. However, we are unable to get growth in LB-Amp (100 ug/ ml Amp) liquid media. Other plasmids have worked with the same set of reagents, plates, and liquid media. But pMX-puro refuses to grow. Please help.
Thanks so much.
Cheers!
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3 replies to this topic
#2
pito
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Posted 26 January 2011 - 02:21 PM
You get growth on plates and then you pick up a colony, inoculate a liquid medium and then it wont grow anymore? Is that the problem?
Anyway, you use twice the amount if amp , maybe you need to try it with 50 to check whether its the amp that is killing your bacterium in the liquid medium or the medium (the fact that its liquid) itself that is bad. (medium itself can also mean the way you store it, temperate, enough oxigen..)
and you are sure the media is the same? you made it toghether with the only differce being the agar? (and the amp after the autoclaving).
Anyway, you use twice the amount if amp , maybe you need to try it with 50 to check whether its the amp that is killing your bacterium in the liquid medium or the medium (the fact that its liquid) itself that is bad. (medium itself can also mean the way you store it, temperate, enough oxigen..)
and you are sure the media is the same? you made it toghether with the only differce being the agar? (and the amp after the autoclaving).
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
#3
toxotuesday
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Posted 26 January 2011 - 03:40 PM
Right.
But would a two fold difference in ampicillin concentration matter?
But the liquid LB media has worked for other plasmids. And we store it in a cold room at 4 degrees C.
The plates and liquid media were made from different batches of LB media, but the same ingredients except the agar.
We make the LB and LB-agar following the protocol below:
LB (Luria-Bertani) Medium
Tryptone 10 g
Yeast extract 5 g
NaCl (58.44) 5 g
H2O to 1 L
Sterilize by autoclaving
For LB/Amp plates, add 15 g agar (1.5% w/v) prior autoclaving. Cool to 50˚C. Add 500 µl of Ampicillin (100 mg/ml) per 500 ml of LB or LB agar for a final concentration of 100 µg/ml.
Thank you so much.
But would a two fold difference in ampicillin concentration matter?
But the liquid LB media has worked for other plasmids. And we store it in a cold room at 4 degrees C.
The plates and liquid media were made from different batches of LB media, but the same ingredients except the agar.
We make the LB and LB-agar following the protocol below:
LB (Luria-Bertani) Medium
Tryptone 10 g
Yeast extract 5 g
NaCl (58.44) 5 g
H2O to 1 L
Sterilize by autoclaving
For LB/Amp plates, add 15 g agar (1.5% w/v) prior autoclaving. Cool to 50˚C. Add 500 µl of Ampicillin (100 mg/ml) per 500 ml of LB or LB agar for a final concentration of 100 µg/ml.
Thank you so much.
#4
pito
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Posted 27 January 2011 - 02:11 AM
Well, twice the amount of amp means you double it.. I mean: its a lot.. Its +100%...
It could make a difference.
But normally it shouldnt..
I would suggest you to make new media: make the liquid and solid toghether, so 2 bottles with agar and 2 without agar. Just to be sure its not the media thats bad.
1 bottle with agar medium: add 100µg/ml, the second one 50µg/100ml, the bottle for the liquid medium: 1 with 100µg/ml and one 50µg/100ml.
USe those 4 to check growth. This way you can rule out the fact that it might be the medium thats been bad (someone could have added to much amp , the medium could be bad , too much or not enough of...).
If you inoculate: do inoculate more then just 1plate and 1 tube (so do not simply inoculate 4 in total: 2 plates, 2 tubes, but inoculate more just to be sure).
Another thing you might want to do: take one of the tubes that you tried to inoculate (but failed) and inoculate it with a bacterium that you are certain of that it will grow at 100µg/100ml ==> you said it worked with other plasmids, so check the tubes.
And you do shake the liquid medium when growing the bacterium at 37°C right?
It could make a difference.
But normally it shouldnt..
I would suggest you to make new media: make the liquid and solid toghether, so 2 bottles with agar and 2 without agar. Just to be sure its not the media thats bad.
1 bottle with agar medium: add 100µg/ml, the second one 50µg/100ml, the bottle for the liquid medium: 1 with 100µg/ml and one 50µg/100ml.
USe those 4 to check growth. This way you can rule out the fact that it might be the medium thats been bad (someone could have added to much amp , the medium could be bad , too much or not enough of...).
If you inoculate: do inoculate more then just 1plate and 1 tube (so do not simply inoculate 4 in total: 2 plates, 2 tubes, but inoculate more just to be sure).
Another thing you might want to do: take one of the tubes that you tried to inoculate (but failed) and inoculate it with a bacterium that you are certain of that it will grow at 100µg/100ml ==> you said it worked with other plasmids, so check the tubes.
And you do shake the liquid medium when growing the bacterium at 37°C right?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
