MiniPrep - (Dec/07/2007 )
I have been trying to isolate plasmid using miniprep home made kit from BL21. I am not able to isolate plasmid at all
Using qiagen kit or any other kit is not an option. I need to make this work. But i am so lost at this point. I have tried everything and anything. I used 2 enzymes for digestion. Nco1 and HindIII with Buffer 2. and i should get 2 bands 5.6kb and 350 bp. But i either get wrong bands or none at all. And i dont even get undigested plasmid as well. Oh By the Way, everyone else in my lab cna use the protocol and get isolation ... even an undergrad volunteer student!!! also, i am a post doc in the lab....
so please help
What do you think the problem might be?
The bacteria were overgrown?
You did not add enough antibiotics?
The bacteria were poorly suspended?
NaOH is not good?
Too much RNA comtamination in your prep?
Additional contaminations that interfere RE?
Make a list, go through it one by one.
They dont call you a Dr. for nothing .
Look into all steps for possibilities as to why the miniprep is not working optimally.
If possible, Try different cells.
After you do the miniprep, do you run some on a gel to make certain the DNA is there? If it isn't, then I have two thoughts: 1. maybe you have a low copy plasmid or 2. perhaps you have contamination in your cells and you're not really growing E. coli. Other bacteria have a different smell than E.coli and won't lyse when you add solution 2 to the miniprep. You can check this visually - your prep should become clear after addition of solution 2.