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Nuclei preparation for nuclear RNA extraction


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#1 NemaToStella

NemaToStella

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Posted 23 September 2013 - 01:42 PM

Hello BioForum members,

I'm currently struggeling with purification of nuclei from tissue culture cells, that are to be used for RNA isolation.

Midway through the protocol, the "nuclei" start clumping together, and I have a feeling this is because of genomic DNA spillage (despite careful resuspension with wide pipett tips etc.) ... Also, by microscopic examination I can't tell whether I have pure nuclei or whether there are still intact cells (in trypan blue stainings, the entire nuclei/cells appear blue).

Does anyone have a protocol that works like a charm that he/she is willing to share with me?

 

In brief, here's what I've done:

1) resuspend cells in swelling buffer (10 mM Tris-Cl, pH7.5; 10 mM NaCl; 3 mM MgCl2), incubate 3 min on ice

2) pellet cells (at this point the pellet clearly looks "swollen")

3) resuspend pellet in 4x pellet volume of lysis buffer (10 mM Tris-Cl, pH7.5; 10 mM NaCl; 3 mM MgCl2; 10% glycerol;

   0.5% NP-40; 0.5 mM DTT)

 

This is when the pelleted material starts clumping together (clusters of nuclei/cells stick together when observed under the scope).

 

Any suggestions are much appreciated!






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