I am getting appritiable growth of bacteria in antibiotic medium. On isolating plasmid by conventional method or by kit I am getting either very little amount of plamids or no plasmids or sometimes I get good amount of pellets but it doesn't dissolve. How to fix it?
Hi
you'd better to reduce final elution buffer to concentrate the DNA
are you sure the pellet is your DNA? It may be protein!
Anyway you can put it in 45 c for about 0.5 hour to dissolve
If you are doing the conventional method please be careful with the Alkaline lysis 2 step. Do not exceed the treatment time. This is crucial and if this step not followed property it can result in loss of plasmid.
What kit are you using? Is this a low copy number plasmid? You can try increasing the volume of your overnight culture to 10 mls. I tried isolating a low copy number plasmid with Qiagen and It gave me good yield.
Also check your antibiotic is not out of date. Reduce the volume of elution buffer.
If you have lots of cells then either the plasmid is not present at a high amount or the isolation is not working. A few things to check:
1. your antibiotic - as mentioned above they do expire, then the cells will lose the plasmid
2. the kit - these also have expiration dates - generally they are good for 1 year after opening IF the bottles are all stored at the proper temperature
3. troubleshoot the kit steps
resuspend your cell pellet really well
mix well as directed for all mixing steps
make sure the lysis and neutralization are working (lysate should be clear, neutralized should be very cloudy/thick)
if this is a column kit, try letting the elution buffer sit 1 min prior to centrifuging
if this is a column free kit, be sure to move the cleared lysate to a fresh tube without any debris carryover (that will look like a pellet)
if this is a column free kit, get a fresh solution of alcohol for the precipitation step (it won't work if the alcohol level is too low)
good luck!
- Badges
- Science topic