3 replies to this topic

[n00b]

Hello!

First of all, I'm new to molecular biology, so many apologies if I'm full of questions. Second, this is something of a stupid question, so apologies again!

I just finished Maxiprepping, and I accidentally eluted in TE instead of EB. I need to send these samples for sequencing, but I know the EDTA in TE prevents the sequencing reactions from working correctly. How do I go about repurifying the DNA in water or EB so that I may send it out for sequencing?

We do have a PCR purification kit (the Wizard SV kit from Promega), can I use that?

Thank you!

[bob1]

You can ethanol precipitate the DNA or you could use the PCR purification kit (check the size cut-off), which is essentially an ethanol/isopropanol precipitation with a glass filter to retain the DNA.

Incidentally, I would have done the sequencing on a much smaller prep (e.g. mini) rather than scaling all the way up to a much more expensive maxiprep, before checking.

[phage434]

In my experience, TE has little or no effect on sequencing reactions, despite warnings to avoid it.  It should not, since the EDTA is only 1 mM, and the amount of DNA in the reaction is typically 10-20% of reaction volume.

[n00b]

All right, thank you both!