6 replies to this topic

[BSM]

Hey everyone,

I am planning on performing some maxipreps using Qiagens EndoFree kit and I had a few questions. The current rotor for our high performance centrifuge fits the 50ml round bottom oakridge tubes. I currently have some new 50ml  polycarbonate and polyproplyene tubes that I will use for my plasmid isolation. The manufactures insist that the fill volume must be at 80% capacity (33.6ml) in order to insure proper performance.

A couple of steps in the maxiprep protocol have you spin in a total volume of 25ml and 5ml(precipitation step) at 15,000 x g. This is well under the 80% capacity. Have any of you ever encountered this problem and what did you do to fix it?

Thanks

Edited by BSM, 23 August 2011 - 05:09 PM.

[leelee]

Are you sure its not 80% or less? That's what I always thought......

[BSM]

No it says 80% for proper centrifugation.

Since I plan on performing transfections + mouse microinjections in the future there is also a centrifuge here that can spin 50ml sterile conical vials, but it max RPM is 5300 (avg. g = 4894 and max g=6871). I guess this may work even though the avg g is not quite 5000 which is what the protocol calls for.

Edited by BSM, 23 August 2011 - 08:05 PM.

[little mouse]

It's true that some tubes need to be filled at  80%, unless they will collapse in the centrifuge.

You need to find other tubes, or to dilute your samples.

[BSM]

little mouse, on 24 August 2011 - 06:06 AM, said:

It's true that some tubes need to be filled at  80%, unless they will collapse in the centrifuge.

You need to find other tubes, or to dilute your samples.

So a 5ml ethanol wash step could be replaced with 20-30ml ethanol without problems?

[perneseblue]

Given that the DNA pellet is rather small, I prefer to do the 70% ethanol wash in a 1.5ml microcentrifuge tube.

Thus, after the isopropanol precipitation, I wash off the DNA pellet with 1ml 70% ethanol and move the pellet into a microcentrifuge tube. Spin at maximum rpm (14 000rpm) for 10mins. Remove the 70% ethanol and repeat the wash again with 70% ethanol.

It is a lot easier to see the pellet in a microcentrifuge tube compared to a 50ml tube.

[Gangwolf]

Yes, you can without problem wash your samples with 5ml of ethanol instead of 20-30 ml because your samples will not dissolve in ethanol.