HELP. Mistakenly added 70% EtOH not 100% in PPT - (Jul/13/2008 )

Dear clever generous wonderful scientist.

Here i am at work on a sunday getting a sequencing reaction cleaned up for tomorrow when I accidentl added 70% EtOH not 100%

Here are the details
1uL Big Dye 3.1
3.5uL 5 buffer
3.2uL SP6 primer (for pGEM vector)
12.3uL DNA

Clean up
5uL 125mM EDTA
60uL 100% EtOH

At this step I added 70% not the 100%.

Leave to incubate at RT for 15 min
30min spin
wash with 70% EtOH
dry pellet

Is this going to eff up my sequencing tomorrow?
Please tell me some good news?

Sincerely
Zena

It shouldn't be a problem -- if you got a pellet and if it was 70% ethanol in water. There's nothing that will precipitate upon addition of 70% ethanol that won't also precipitate upon addition of 100% ethanol, so there's not going to be anything there you wouldn't have had anyway, and if your DNA also precipitated when the 70% ethanol was added, you're fine.

If I read correctly het added 70% insted of 100% in the precipitation mixture. When we do precipitation of sequencing we get a final concentration of 63% of Ethanol (by addition of 100%), roughly the minimum in which DNA is not soluble.

By the numbers given here, adding 60 µl of 70% ethanol will not get your final % of alcohol above 63%, so I fear your pellet will be lost.

you can add now the 100% ethanol to precipitate your dna, just calculate the correct minimum volume.

When we sequence, we never actually see a pellet, but proceed and get nice sequences. In our protocols it also says "dry pellet" even if it's invisible (from the fact that we get nice electropherograms there has to be a pellet).
To visualise pellets, one could resort to using "pellet paint". To avoid mistakes, I make a bigger stock solution with "precipitation mixture" (contains everything so I just have to add this to my sequencing reaction) making mistakes on important samples less likely (if it worked the first run, it will work for all next runs).

I am afraid that you have to do the sequencing once again. It happened with me in the past time and I corrected the concentration by adding 99% Etho but it did not work.

Relax and start again

Mahdijp
.

Ding ding ding!!!!!!!!!!!!!

Mahdijp had the right answer!!!! :-)

I tried the "pilot" sequences .. and it was all noise with a signal ranging from 1 - 6 ..

In any case, I repeated the clean-up using the right concentration of EtOH this time ;-)
And the sequencing is perfect.
Actually out of the 96 sequences I got about 10% failure .. which isn't bad since I used the R.E.A.L extraction kit.

Anyway, thanks everyone so so much for your comments and suggestions.
Sure initially my sequences didn't work but at least I wasn't stressing about them in the mean time.

Cheers

- Zena