During my plasmid purification with MN midiprep kit I loose my pellet after washing with 70% EtOH and centrifuge at 15,000 5min RT.
I clearly see a huge amount of precipitate after using isopropanol and centrifuge at 15,000xg 30min 4C. Removing the isopropanol goes well, pellet stays on the side of the tube.
After I add the 70% EtOH pellet disappers and I see nothing after centrifuge at 15,000xg 5min RT.
Any idea?
Hi there,
Are you sure there is no DNA at the end? Nothing visible doesn't mean there is actually nothing in the tube. The crucial thing when preparing DNA is to be careful when removing supernatant because sometimes pellet just goes with it. If i were you I would run the prep until the end and then measure UV absorbance.
Thanks for the answer Dominique!
Last time I did measurement of UV absorbance but there was a tiny amount of DNA in it.
Normally it should be visible after washing and centrifuge, right?
Just adding one extra point to Dominique's suggestion, the purpose of 70% alcohol wash is to remove the impurities (mainly salts) that get precipitated along with DNA during precipitation steps. DNA remain insoluble in 70% alcohol, but the salt gets dissolve and comes in the soluble phase. So before alcohol wash, the pellet can be seen as a white visible dot, but after the wash it become small, and transparent so can't be easily seen and get camouflaged with the bottom of the tube. So follow what Dominique has suggested.
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