LB + X-gal plates + Amp plates - (Jan/24/2006 )
Hi all,
I am doing a ligation with TOPO T/A cloning kit and went to make some LB plates with X-gal and Ampicillin. I accidentally added too much ampicillin, so rather than having 100 ug/ml I have 200 ug/ml. Does anyone know if I an use the plates? I am transforming the ligation mix into Top10 cells. Thanks.
M.
Hi, don't know about that amount... used to take old Amp plates(made up as 100ug/ml) after 2-3 mos in 4C and add 50-75ug/ml amp to them and use them they worked fine with JM109 but have no way to know how much amp activity was left on them after storage...
I would say if it is a precious sample then wait...
If you have already transformed then you could also plate one plate say 100ul of 1ml transformation then store the rest fo the transformation at 4C until tomorrow to test (would also give time to make up new plates...) wouldn't risk that if it is a very precious sample (like it took a long time to prep or you don't have much left) but if you have more ligation left or all of the reagents you need then I might try it... look at it like this if it works you save a day if not you only lose one day that you were gonna lose anyway...
good luck!!!
Yeah I agree, its no biggie but I thought I could save the plates. Its one of those things when you have 10 other items in your mind that you make a stupid mistake! I'll give it a go and let everyone know how it goes.
I would say if it is a precious sample then wait...
If you have already transformed then you could also plate one plate say 100ul of 1ml transformation then store the rest fo the transformation at 4C until tomorrow to test (would also give time to make up new plates...) wouldn't risk that if it is a very precious sample (like it took a long time to prep or you don't have much left) but if you have more ligation left or all of the reagents you need then I might try it... look at it like this if it works you save a day if not you only lose one day that you were gonna lose anyway...
good luck!!!
I am doing a ligation with TOPO T/A cloning kit and went to make some LB plates with X-gal and Ampicillin. I accidentally added too much ampicillin, so rather than having 100 ug/ml I have 200 ug/ml. Does anyone know if I an use the plates? I am transforming the ligation mix into Top10 cells. Thanks.
M.
I suggest you keep those plates around 45-50 degree for 24-48 hrs and that wil reduce the activity of Amp.
FN
Hi all,
I am doing a ligation with TOPO T/A cloning kit and went to make some LB plates with X-gal and Ampicillin. I accidentally added too much ampicillin, so rather than having 100 ug/ml I have 200 ug/ml. Does anyone know if I an use the plates? I am transforming the ligation mix into Top10 cells. Thanks.
M.
I suggest you keep those plates around 45-50 degree for 24-48 hrs and that wil reduce the activity of Amp.
FN
Yes I agree to keep plates at 45-50 degree for 24-48 hours to reduce the activity of amp.
I really recommend you to quickly prepare fresh plates. It would take you maybe 3 hours (including autoclaving, plating, cooling down) or ask another person in your lab or the lab close to yours if they some extra Amp. plates not older than 2 weeks.
If you already transformed the bacteria with your ligation product because is in recovery medium (LB without antibiotics) keep it either at 4C until ready to plate
or
add some liquid LB with 100ug/ml amp. and before plating spin down the cells resuspend them in 100ul or 200ul medium and spread on the fresh plate.
I wouldn't risk my liagation product in plates with 200ug/ml amp.
And actually I don't think is a good idea to use colonies grown in there.
because you might contribute to create some non transformed bacteria resistant to antibiotic and later on in the lab you might need to start using 200ug/ml as standard instead of 50 or 100 ug/ml.
I know this might sound strange but actually it happened in our lab. We were using only 50ug/ml Amp in our lab but one person was using 100 ug/ml. After a while we all had to change to the higher conc of antibiotic 100ug/ml.
Maybe someone can add something to this, explaining how it happens?
FN
i would not do that for so long time as the resulting amp will not be enough for proper selection.
i use 200µg/ml amp in plates for a month as i did the same mistake as you.
All is as usual.
I agree with Fred; use them up
If I am setting up to do some mini-preps, and I know it will be a longer overnight (won't be in till late or something) I deliberately grow the colonies in 200ug/mL to maintain proper selection. might be different on solid media, but I think you will still get growth IMHO
thanks guys. I will try using the plates, I hate waste!!. If it fails I will simply re-do the transformation.