Accidently used Elution buffer instead of wash when purifying PCR products and plasmids in Qiaquik columns. Currently, I was wondering if anybody knew if it was possible to recover the DNA, or do I need to start over?
Yes, I had a student who made this mistake once. It depends on the volume you added to the column. If it was 50 uL or less, simply add the first buffer (QC or equivalent) at a 3:1 ratio and start over, and you can expect a decent recovery of DNA. If instead you eluted in a large volume, you can try to ethanol precipitate as Luiza suggested. However, if the total amount of DNA is low (
How valuable are your samples? From experience it is probably better to cut your losses and start again. You need to be sure of your end product; personally if I knew something had gone wrong I would be sceptical of down stream use/results and you could end up wasting more time with repeats if they didn't work. if you can salvage the samples it wouldn't do any harm to repeat anyway. I guess you could either precipitate the DNA out of the elution buffer somehow and then wash it. or just continue and hope you still have DNA bound to the column that hasn't eluted. sorry I can't be of anymore help. good luck.
ps the companies are usually good to contact for tech support.
I never tried, but you can preciptate your dna from the elution buffer using 2 volumes of EtOH 100% or 0,7 volumes of Isopropanol. Mix the alcohol with your sample, centrifuge for 30 min on the highest rpm you have and pray for a pellet to appear. If it doesn't, try to elute it anyway, sometimes it works. Good Luck.
Hi Joe,
I am quite sure the trick of the elution buffer is to change the pH and therefore release the PCR products from the column. Then, if you change the pH back to the fixation buffer (check in the same kit) and put your samples back trough the columns, you should recover your precious PCR products. You can also try adding a couple of volumes of the fixation buffer to your already eluted samples and go trough the columns again.
Good luck!, and let us know how it works!
Yes, I had a student who made this mistake once. It depends on the volume you added to the column. If it was 50 uL or less, simply add the first buffer (QC or equivalent) at a 3:1 ratio and start over, and you can expect a decent recovery of DNA. If instead you eluted in a large volume, you can try to ethanol precipitate as Luiza suggested. However, if the total amount of DNA is low (
I did the same mistake with the Giga prep protocol. Then I diluted the eluted fractions with QC buffer (1:4.5) and start over again. I managed to save most of it. In the end, I could actually see a nice DNA pellet after isopropanol precipitation.
My samples were around 1250-3180ng/µl.
A260/A280 was from 1.88-1.9
A260/A230 was from 2.28-2.39
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