nuclear extraction problem - nuclei gets too clumpy - (Oct/07/2010 )
Hi, I'm having some problems with making nuclear extract from NK92 cells (lymphocytes, suspension). The problem is that after I collect nuclei and try to resuspend in my nuclear extract buffer, I cannot get it to resuspend and the prep turns clumpy and gooey. I suspect I've busted the nuclei and released genomic DNA, but there is no detergent in extraction buffer so I'm not sure how it's possible. Here's my protocol
1. wash ~1x10^6-1x10^7 cells x2 in cold PBS
2. pellet cells 5k rpm 5min 4C
3. Lyse cells using 500ul of lysis buffer (Tris/EDTA, 60mM KCl, 1mM DTT, protease inhibitor tablet, 0.1% NP-40) for 5 min on ice
4. Pellet nuclei at 2.5k rpm 4 min 4C
5. Wash with buffer (same as lysis buffer but no NP-40) once - still not clumpy, easily resuspended at this point
6. pellet nuclei at 2.5k rpm 4 min 4C
7. Resuspend in 100ul nuclear extract buffer A (Tris/EDTA, 420mM NaCl, 1.5mM MgCl2, 25% glycerol) OR buffer B (same as A, but with 800mM NaCl instead of 420mM) - after addition, pellet becomes clumpy and gooey
8. ice for 20 min, spin down at max speed 6min 4C and take supernatant
Also, when I take the supernatant, I end up with less than 100ul. I think I'm losing a fraction to being trapped in the clump/goo.
Any insights much appreciated...
Ben
laichiehmin on Thu Oct 7 21:51:08 2010 said:
Hi, I'm having some problems with making nuclear extract from NK92 cells (lymphocytes, suspension). The problem is that after I collect nuclei and try to resuspend in my nuclear extract buffer, I cannot get it to resuspend and the prep turns clumpy and gooey. I suspect I've busted the nuclei and released genomic DNA, but there is no detergent in extraction buffer so I'm not sure how it's possible. Here's my protocol
1. wash ~1x10^6-1x10^7 cells x2 in cold PBS
2. pellet cells 5k rpm 5min 4C
3. Lyse cells using 500ul of lysis buffer (Tris/EDTA, 60mM KCl, 1mM DTT, protease inhibitor tablet, 0.1% NP-40) for 5 min on ice
4. Pellet nuclei at 2.5k rpm 4 min 4C
5. Wash with buffer (same as lysis buffer but no NP-40) once - still not clumpy, easily resuspended at this point
6. pellet nuclei at 2.5k rpm 4 min 4C
7. Resuspend in 100ul nuclear extract buffer A (Tris/EDTA, 420mM NaCl, 1.5mM MgCl2, 25% glycerol) OR buffer B (same as A, but with 800mM NaCl instead of 420mM) - after addition, pellet becomes clumpy and gooey
8. ice for 20 min, spin down at max speed 6min 4C and take supernatant
Also, when I take the supernatant, I end up with less than 100ul. I think I'm losing a fraction to being trapped in the clump/goo.
Any insights much appreciated...
Ben
Ben,
I am having the same problem. Did you ever find a solution or determine what was causing it? Thanks.
jetzs on Thu May 5 20:47:21 2011 said:
laichiehmin on Thu Oct 7 21:51:08 2010 said:
Hi, I'm having some problems with making nuclear extract from NK92 cells (lymphocytes, suspension). The problem is that after I collect nuclei and try to resuspend in my nuclear extract buffer, I cannot get it to resuspend and the prep turns clumpy and gooey. I suspect I've busted the nuclei and released genomic DNA, but there is no detergent in extraction buffer so I'm not sure how it's possible. Here's my protocol
1. wash ~1x10^6-1x10^7 cells x2 in cold PBS
2. pellet cells 5k rpm 5min 4C
3. Lyse cells using 500ul of lysis buffer (Tris/EDTA, 60mM KCl, 1mM DTT, protease inhibitor tablet, 0.1% NP-40) for 5 min on ice
4. Pellet nuclei at 2.5k rpm 4 min 4C
5. Wash with buffer (same as lysis buffer but no NP-40) once - still not clumpy, easily resuspended at this point
6. pellet nuclei at 2.5k rpm 4 min 4C
7. Resuspend in 100ul nuclear extract buffer A (Tris/EDTA, 420mM NaCl, 1.5mM MgCl2, 25% glycerol) OR buffer B (same as A, but with 800mM NaCl instead of 420mM) - after addition, pellet becomes clumpy and gooey
8. ice for 20 min, spin down at max speed 6min 4C and take supernatant
Also, when I take the supernatant, I end up with less than 100ul. I think I'm losing a fraction to being trapped in the clump/goo.
Any insights much appreciated...
Ben
Ben,
I am having the same problem. Did you ever find a solution or determine what was causing it? Thanks.
Hola probably the viscosity of the sample and difficulty to load is due to the presence of whole DNA in the sample. I have a sonicator with a fine needle wich lead to broke DNA in 3 sec and viscosity disappears. Other possibility is load the hot sample directly from the heater, it doesn´t broke DNA but you will have less difficulties to load the sample. Buena suerte
protolder,
Thanks for your reply. Initially I was using a protocol that called for sonication (solutions from scratch). We decided to try a Nuclear Extraction Kit because of the gooey pellet problem. I am not sure I trust the results of the extraction with a gooey pellet?
I did the same experiment in two different cell lines and extracted them with the kit at the same time. One had goop and one did not.
I am baffeled.
The only thing I can think that would may a difference, is the confluence of the cells. The one that gooped up was about 60% confluent upon harvest. The one that did not goop up was about 95% confluent.
If the goop is DNA that would make sense--more of the tertiary structure of DNA would be unwound in actively growing cells and may allow the DNA strands to associate under the conditions of nuclear extraction. Any thoughts on this are appreciated.