Centricon/microcon question - Can these spin columns expire? (Dec/13/2007 )

I tried to purify cesium-banded DNA today. I butanol-extracted the ethidium bromide 3 times, and then concentrated the DNA sample (~3 micrograms environmental genomic DNA) in a spin column. The final eluate (200 microliters) had no detectable DNA. I should have been able to detect DNA even if I had lost 60% of it (it would still be at 5 ng/microliter, which I should have been able to detect on the nanodrop.) The nanodrop reading was at best 1.5 ng/microliter, a reading which I have zero confidence in and which would represent a loss of ~90% of my original DNA.

I don't know where I could have lost it. The spin columns are very old. Is it possible they developed microfractures and that the DNA flowed through the filter? I used amicon centricon 100s for most samples, and microcons for a few samples (we ran out of the centricons).

Thanks for any helpful ideas.

Patty

---

I've lost many samples using old Microcons. Perhaps the membrane just deteriorates after a while. I don't know. But I am reluctant to use Microcons for important samples.

---

yes, spin columns can and do expire. After their expiry date, the columns start to go.... not all at once and there seems to be somekind of batch differences

---

i use microcon quite frequently and till now I never had problems. I use YM-100 (blue) and YM-30 (white).
The centrifugation speed is very important especially with YM-100 which should not be spinned at more than 100g for 12 minutes. I pre-clean the filter with 200ul of sterile water and spin it. Then I add sample + 200ul of sterile water and spin. before inverting the filter I add a small volume of water and leave it for 5minutes. Then invert and spin at 1000g for 3 minutes.
Lately I started using the YM-30 and I found these better since I can spin them at a much higher speed up to 14,000g.
I used microcon to clean PCR products from attached nucleotides etc although for this purpose I also use a product from GENETIX which is also cheaper.
I find microcon very good to cleeen products from buffers between restriction digests and for DNA that I need to use for cloning.
hope this helps

---

Thank you for the feedback.

I believe I will check the concentration with pico green, which is much more sensitive than the nanodrop for quantitation. At least it might give me some confidence in the actual concentration of recovered DNA.

I am undecided as to whether to switch back to EtOH precipitations for future purifications, or just make sure the columns are fresh, after this experience.

I spun the columns at 1000g (100g seems very low!) as per the protocol, and did three washes. Perhaps I should cut back to two washes ...

---

I've got a sub-question to this:
The Microcon filters are preserved in glycerol. If you don't rinse the glycerol from the filter before use - has anyone seen any effect downstream (eg PCR inhibition)?
Right now I use a lot of Microcons but I'm thinking for my less concentrated samples glycerol washed through to final product might just be an issue.

Anyone got any experience?

---

You can prewash the microcon filters to remove glycerol, which is good practice for any of the filters.

---

FWIW, we've had trouble when we've tried to pre-wash the filters. What happened most recently was that we tried to pre-wash the centricon 100s (remember, they are old) with water. It didn't go through. At all. We spun the column for over an hour. The water could not get through the filter. If we skip the water wash and load the sample directly, the columns appear to work OK (ie the sample flows through in a ten minute spin.)

I didn't want to mention this in my original post.

Update: the picogreen confirms the nanodrop readings, so I have more confidence that I've got DNA, just not very much DNA. At least it is clean.

---