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seqance 05-30-2018 01:53 AM

DNA bead purification
 
Hi all,
I am new in NGS library prep,when i purified DNA with beads i lost most of my input.
How to perform DNA purification using bead whithout losing material?
Could someone help?
Many thanks

ATϟGC 05-30-2018 05:40 AM

I find it can be hard to get more than 80-90% recovery and I sometimes get as low as 50%.

Here are a few suggestions you can try for improving recovery:

-Do not dry the beads too long. If you see cracks forming in the bead pellet then they have been dried too long and elution of the DNA from the beads can be difficult. I try to dry the beads to the point they look like a wet or moist clay and then add the elution buffer. Over-drying the beads is a common cause of poor recovery.

-Elute at 50-60 degrees Celsius for 30 minutes or longer. You may also leave them at 4 degrees for a few days to help with elution.

-Try a custom elution buffer containing tween-20 such as: 10 mM tris-HCl pH 8.0, 0.1 mM EDTA pH 8.0, and 0.1-0.5% tween-20 v/v.

Please note that I have not rigorously tested these modifications in a systematic manner and they might not help significantly.


It would be helpful to know what size range of DNA you are trying to purify, what beads you are using and a description of your protocol.

seqance 05-31-2018 01:10 AM

Hi AT/CG,
Thank you for your reply.
It is my first library prep, beads were over dried because i saw cracks.
I ll try again and let beads dry for less time.

ATϟGC 05-31-2018 04:28 AM

You are very welcome. Sounds like over-drying is likely your main problem. If you are worried about ethanol contamination and want to minimize drying time you can remove as much ethanol as you can from the tubes, briefly spin the tubes to collect the liquids and beads at the bottom, then move the paramagnetic beads to the side of the tube with a neodymium magnet and collect the remaining liquids with a 10-20 ul pipette tip. After this, a minute or two of drying time should allow virtually all the ethanol to evaporate and the beads should look like moist/wet clay but not cracked.

seqance 05-31-2018 05:08 AM

I'll try it next week. Thank you AT/GC.

ECO 05-31-2018 02:52 PM

If you "over dry", just "over elute" and make sure to pipet to break up the chunks and leave it to elute for a much longer time than usual (>15 minutes).

seqance 06-01-2018 04:55 AM

Thank you ECO. would you suggest me 'websites or other ' to learn about nsg?


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