Problem with centrifuging the sample - (Apr/30/2011 )

During my maxiprep, I wanted to precipitate the crude lysate with isopropanol before loading on the column (Qiagen maxiprep). So, I added room temp isopropanol upto the top of 50 ml BD Falcon polypropylene tube (about 20 ml per tube)which already had about 30 ml cleared lysate.I made sure there was no overflow or leak and mixed the samples by inverting the tubes several times. After this, when I centrifuged the tubes at 4000 rpm for 1 hour at 4 degree C in J6-MJ Beckman centrifuge with JS-4.2 rotor), some tubes broke completely and liquid spilled all around the bucket and some tubes had cracks around the wall. Interestingly, it happened again when I repeated the same procedure next day-this time using 48 ml total volume instead of filling to the top. And, I know this does not happen when I try to centrifuge the column flow-through (about 15 ml) with isopropanol (about 10.5 ml), using the same condition. Do you guys have any similar kind of experience? Any thought on what's going on? The maximum centrifugal speed tolerated by these tubes in at least 6000g under standard condition and the 4000 rpm I am using in this rotor is well below that limit? May be I should not fill up the tubes more than 35-40 ml? or may be temperature is the factor?

Thanks,
Manish

Are these the conical bottom 50 ml centrifuge tubes ("Falcon tubes")? These are usually good to about 8000 x g. I suspect your rotor is not supporting the conical bottom correctly. Have you looked at the bottom of the insert?

Yes,these are 50 ml Falcon conical tubes. I know that, in theory, these tubes should be ok upto at least 6000g. Intriguingly, when I add 15 ml lysate plus 11 ml isopropanol and centrifuge at 4000 rpm, the tubes look fine (unlike filling up the tube with crude lysate plus isopropanol). So, I don't think rotor is causing the problem. May be it is the volume in the tube or something in the crude lysate or combination of both is reducing the maximum g value that these tubes can withstand? just a thought...

Manish

It could still be the rotor - if there isn't enough mass in there to affect the tube shape when spinning, then they won't crack.

As Phage434 says you should check the shape of the buckets in the rotor, if they are not conical bottoms that fit the tubes, then you need to find appropriate inserts for them.

I checked the rotor as you suggested. The buckets have green colored Beckman Adapters, Model 345371 with black pads (345369)-they have conical shaped structure at the bottom for holding the 50 ml conical tubes. But, may be you are right- the mass of 50 ml lysate to too much (probably too heavy) to affect the shape of tubes during spinning.. would appreciate any other thoughts..

That sounds like they should work well. Another possible issue is side support of the tubes. Is the diameter of the hole well matched to the diameter of the tube?

It happened with me in another situation, problems may be the conversion between G and RPM. Check if 4000 RPM < 6000 G for your centrifuge (refer to the catalogue). Another issue is that if you are using tubes you autoclaved or used before so that this will make the tube weaker. I suggest you to use Nalgene tubes which can go up to 50000 G.