miniPrep troubleshooting - (Oct/28/2009 )
What should be happen if the cell culture stay in the room temperature for more than 8 hours without rotation after 37C overnight incubation when we do the miniPrep? Does it infect the miniprep result? It looks like normal when I run a gel after digestion. But it always get very short sequence when I use this DNA for sequencing. Any explanation? Thanks a lot.
subtelo on Oct 28 2009, 07:51 AM said:
Maybe it's your sequencing reaction that is failing? If the digestion patterns are OK, then I would think that the plasmid is intact. You can try and do a PCR on the fragment you want to sequence and check the length of that.
Make sure you are sequencing the proper amount of DNA. Too much template (or too little) in a sequencing reaction will give the results you see. Run the DNA on a gel and compare band strengths with a ladder of known concentration. This is more trustworthy in eliminating confusion of RNA and genomic DNA than a simple spec reading.
your plasmid may be supercoiled. you need to relax the supercoil prior to sequencing.
mdfenko on Oct 29 2009, 10:17 AM said:
Thank you all for the inputs. They are valuable.
I am not sure how to relax the supercoil? Heat it or something else?
And I also want to know if the cell will be died if stay in the room temperature for more than 8 hours without air. Does it infect the sequencing if the DNA extraction is done with the died cell? Thanks.
heat at 96C for 1-3 minutes or 85C for 5 minutes in water. this will "nick" the plasmid and relax the supercoil.
or you can cut the plasmid (away from the insert) to linearize it.
subtelo on Oct 28 2009, 11:51 PM said:
Hmm... I have never tried this before. I always miniprep my cultures the next morning.