I have Whatman UNIFILTER microplates used for DNA extraction from Arabidopsis thaliana and I would like to know if there is a way to clean and re-use them (they are very expensive).
Strong acid reliably breaks down DNA.
I have been re-using Qiagen silica membranes with good success. You can do that as long as you are not intending to generate presence-absence data (e.g. AFLP or similar) or detecting presence/absence of a species or similar. Hence, if you are going to sequence a genomic region or amplify microsatellites from genomic DNA, cleaned-up membranes are OK to use. Be aware though that the protocol to clean up takes a bit of time and there are probably cheaper membranes around than the one you are using. Macherey Nagel and Pall Life Sciences provide fairly cheap membranes.
Whether or not your membranes tolerate 1N HCl you would have to check in the manufacturer's specifications. Not all kinds of membranes do.
We are using the following protocol:
Wash membrane in plenty of warm water. Remove water, let dry.
Soak membrane overnight (or longer) in 1N HCl. Be extremely careful (use gloves and goggles, protective clothing) while handling 1N HCl because this is a strong acid!
Remove the acid (reuse it), carefully rinse membrane with plenty of deionized water (don't splash!) Fill membrane with 1 ml of ddH2O, spin at 3000 rpm, discard flow-through. Repeat. To neutralize, apply 500 uL of wash buffer to each well, spin 5 minutes to dry the membrane.
Wash buffer (Elphinstone, Hinten, Anderson, & Nock, 2003)
Substance Quantity for 1L
50% Ethanol 500 ml of 96 % ethanol
10 mM Tris 10 ml of 1 M solution, pH=7.4
0.5 mM EDTA 0.14612 g
50 mM NaCl 2.922 g
If you take your work time into consideration, it may be cheaper to buy the membranes.
Good luck
Wouldn't recommend it!!!. Specially if you're going to use the DNA for PCR. You're investing in contamination. Repeating everything from the scratch is more expensive.
Thank you for the answer!but I do not need DNA for PCR, I just want to understand if my DNA extraction protocol is efficient or not. To make the trials I do not want to waste plates and i was wondering if I can re-use them even if contaminated from scratch DNA.
You must not. Once you have used them once the poreof the filter are not reliable any longer and you will contaminate any second filtration product.
The type of plate (plastic), filter type, and pore size are important. Finer pore sizes clog after 1 use and are unreliable. Courser pore sizes with non-binding filter membranes (like those used for sephadex column purification) can safely be reused several times. I suspect you are using plates with a charged filter membrane (and small pore size) that should be discarded after a single use.
Not only is it not recommended, it will not be a very good way of generating clean and trusting data.
Strong acid reliably breaks down DNA.
I have been re-using Qiagen silica membranes with good success. You can do that as long as you are not intending to generate presence-absence data (e.g. AFLP or similar) or detecting presence/absence of a species or similar. Hence, if you are going to sequence a genomic region or amplify microsatellites from genomic DNA, cleaned-up membranes are OK to use. Be aware though that the protocol to clean up takes a bit of time and there are probably cheaper membranes around than the one you are using. Macherey Nagel and Pall Life Sciences provide fairly cheap membranes.
Whether or not your membranes tolerate 1N HCl you would have to check in the manufacturer's specifications. Not all kinds of membranes do.
We are using the following protocol:
Wash membrane in plenty of warm water. Remove water, let dry.
Soak membrane overnight (or longer) in 1N HCl. Be extremely careful (use gloves and goggles, protective clothing) while handling 1N HCl because this is a strong acid!
Remove the acid (reuse it), carefully rinse membrane with plenty of deionized water (don't splash!) Fill membrane with 1 ml of ddH2O, spin at 3000 rpm, discard flow-through. Repeat. To neutralize, apply 500 uL of wash buffer to each well, spin 5 minutes to dry the membrane.
Wash buffer (Elphinstone, Hinten, Anderson, & Nock, 2003)
Substance Quantity for 1L
50% Ethanol 500 ml of 96 % ethanol
10 mM Tris 10 ml of 1 M solution, pH=7.4
0.5 mM EDTA 0.14612 g
50 mM NaCl 2.922 g
If you take your work time into consideration, it may be cheaper to buy the membranes.
Good luck
When working with DNA or RNA samples especially for PCR but not only, must have a very clean area and use new devices, bear gloves, take care of debris, hair. Any minor trace may interfere the results
Try exactly as Silke suggested. We have been also reusing DNA purification columns this way (even without wash buffer as the last wash).
Hi,
What's the cat# of the 96-well Whatman glass fiber microplates you used for DNA extraction. There are many Whatman glass fiber plates. I don't know which one are the best for DNA binding and plant DNA extraction.
Thanks.
Shunxue
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