Life of a Lab Rat

I don't know if this is a good idea, whether people will like it, or even if I have the energy/inspiration to keep it up, but I thought a 'Weekly Protocol' feature might work.

Let's see how we go. I'll start with this one, for making splicing competent nuclear extracts. Cobbled together from a number of different sources, it's surprisingly easy and straightforward.

Printable: http://blogs.usyd.edu.au/labrats/pdfs/Nuclearextract.pdf

Quick and Easy Nuclear Extracts

Notes

1. The method appears to work best with fresh (not frozen) cells
2. This will yield a splicing-competent extract in theory; in practice it might take you a few goes to obtain an extract that works
3. The volumes depend on your initial packed cell volume. I’ve given examples for a 1 ml pellet, which I obtained from five T175s of sub-confluent (i.e. rapidly growing) HEK293 cells.
4. Add 1 mM DTT, PMSF, leupeptin/‘Complete’ etc. (‘goodies’) to HLB and NEX just before use.

Reagents

Hypotonic Lysis Buffer (HLB)

• 1.5 mM MgCl₂
• 10 mM KCl
• 10 mM HEPES-KOH pH 7.9

Extraction Buffer (NEX)

• 1.5 mM MgCl₂
• 0.42 M NaCl
• 0.2 mM EDTA
• 25% v/v glycerol
• 20 mM HEPES-KOH pH 7.9
• 10% IGEPAL (NP-40) in water

Buffer D

• 100 mM KCl
• 0.2 mM EDTA
• 20% glycerol
• 20 mM HEPES-KOH pH 7.9
• 1 mM DTT, PMSF added fresh

Method

1. Harvest cells with trypsin in the usual way and wash twice with PBS (use 5 minute, 400 x g spins). Estimate the volume of the packed pellet.
2. Gently resuspend the cell pellet in 4 volumes cold HLB + goodies (i.e. 1 ml cells + 4 ml HLB). Sit on ice for 15 minutes. Check under microscope that cells have swollen.
3. Add IGEPAL to 0.6% final (300 µl of 10% stock), mix and incubate on ice, 5 minutes.
4. Centrifuge for 5 minutes at 400 x g, 4ºC.
5. Resuspend the pellet (nuclei) in 4 volumes (4 ml) NEX + goodies. Mix at 4ºC for 15 minutes.
6. Pellet debris by centrifugation at 10k x g for 30 minutes at 4ºC.
7. Dialyse against 0.5 l appropriate buffer overnight (e.g. Buffer D for splicing extract), and against fresh buffer for 4 hours, all at 4ºC.
8. Centrifuge for 20 minutes to remove any cloudiness.
9. Freeze drop-wise directly into liquid nitrogen and store at -80ºC.