miniPrep troubleshooting (View forum version)



subtelo

Posted 28 October 2009 - 07:51 AM

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.

elpollodiablo

Posted 29 October 2009 - 01:39 AM

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.


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.

phage434

Posted 29 October 2009 - 04:07 AM

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.

mdfenko

Posted 29 October 2009 - 06:17 AM

your plasmid may be supercoiled. you need to relax the supercoil prior to sequencing.

subtelo

Posted 29 October 2009 - 06:54 AM

your plasmid may be supercoiled. you need to relax the supercoil prior to sequencing.

Thank you all for the inputs. They are valuable.

I am not sure how to relax the supercoil? Heat it or something else?

subtelo

Posted 29 October 2009 - 07:00 AM

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.

mdfenko

Posted 29 October 2009 - 07:01 AM

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.

jiajia1987

Posted 29 October 2009 - 04:53 PM

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.


Hmm... I have never tried this before. I always miniprep my cultures the next morning.