plasmid extraction and antibiotics matters... - (May/21/2005 )

hi all...

my colonies are perfectly grown on LB-Kanamysin-Ampicillin agar (coz the putative plasmid contained that two antibiotics)..problems came after extracting plasmid via kit (from Amersham),i didnt get any plasmid at all..why is that?is there any way that i can figure out which colony got the right plasmid and which aren't?
hmm..i wonder if my antibiotics are no longer active..can anyone tell me how we could simply know that our antibiotics still working or not?
im tired doing clonning works repeatedly..sigh..help please?thanks..

Hi,

There are several possibilities. yes, first check your antibiotics. How old are they? Did you use the correct amount? Did you innoculate the colonies in 1-5 ml LB medium with antibiotics and culture them o/n with shaking and use the o/n culture for plasmid prep? Is the plasmid a low copy number one? How did you know there is no plasmid?

A quick way of checking if the colonies contain positive plasmid is to do a colony PCR. Prepare your PCR mixture as usual, use a yellow tip to pick up a colony, then dip the tip into your PCR mixture and remove it. Run your PCR.

Good luck.

Hi,
I have had similar problems in the past...Try some of these..

1. Make sure that you have added antibiotics at each step. Plating, amplyfying etc.. I also hope you guys make alliquots of the antibiotics and store them at -20. The antibiotic dilutions do not tolerate repeated freeze-thaw cycles.

2. A collegue of mine did not get any plasmid as she had added too much antibiotic..therefore check for the optimal concentration.

3. I have also been told that antibiotics can get degraded if added to the agar when its too hot or when the plates are stored at 37 degrees for too long (>48h). I dont know how true this is...

4. Make sure that your kit is new. Most of these have an expiry date. If it repeatedly does not work..then you may want to talk to the manufacturer for a replacement. Most of them do...

5. Also make sure that you have loaded the optimal amount of luria broth in the columns. you have to also consider the plasmid copy number. If it is a low copy you may need to load more culture.
Hope this helps.

good luck...Keep up the spirits..It will work finally.
Experience speaks
p...

Hi,
I have had similar problems in the past...Try some of these..

1. Make sure that you have added antibiotics at each step. Plating, amplyfying etc.. I also hope you guys make alliquots of the antibiotics and store them at -20. The antibiotic dilutions do not tolerate repeated freeze-thaw cycles.

2. A collegue of mine did not get any plasmid as she had added too much antibiotic..therefore check for the optimal concentration.

3. I have also been told that antibiotics can get degraded if added to the agar when its too hot or when the plates are stored at 37 degrees for too long (>48h). I dont know how true this is...

4. Make sure that your kit is new. Most of these have an expiry date. If it repeatedly does not work..then you may want to talk to the manufacturer for a replacement. Most of them do...

5. Also make sure that you have loaded the optimal amount of luria broth in the columns. you have to also consider the plasmid copy number. If it is a low copy you may need to load more culture.
Hope this helps.

good luck...Keep up the spirits..It will work finally.
Experience speaks
p...