Mar 21, 2018
- Xavier Contreras1,
- Rosemary Kiernan1
- 1Institut de Genetique Humaine, UMR9002, Montpellier, France
External link: https://doi.org/10.1371/journal.ppat.1006950
Protocol Citation: Xavier Contreras, Rosemary Kiernan 2018. High Salt Nuclear Extract Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.kh2ct8e
Manuscript citation:
Contreras X, Salifou K, Sanchez G, Helsmoortel M, Beyne E, Bluy L, Pelletier S, Rousset E, Rouquier S, Kiernan R, Nuclear RNA surveillance complexes silence HIV-1 transcription. PLoS Pathogens 14(3). doi: 10.1371/journal.ppat.1006950
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 31, 2017
Last Modified: March 24, 2018
Protocol Integer ID: 8474
Keywords: nuclear extract, Dignam, transcription
Abstract
Adapted from Dignam JD, Lebovitz RM, Roeder RG. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11(5):1475-89. PMID:6828386
Guidelines
The protocol is suitable for HeLa S3 cells. Parameters should be adapted for other cell types.
Before start
Prepare the following buffers and store at +4°C:
| PMSF 0.2 M: 3.48 g /100 ml DMSO | ||||
| TGME: Glycerol Tris, pH 7.9 MgCl2 EDTA water | Stock 100 % 0.5 M 1 M 0.5 M | Final Conc 50 % 50 mM 5 mM 0.1 mM | Vol (for 1 l) 500 ml 100 ml 5 ml 0.2 ml to 1 l | |
| Low Salt Buffer: Tris, pH 7.3 Glycerol MgCl2 EDTA pH 8 water | Stock 1 M 50 % 1 M 0.5 M | Final Conc 20 mM 12.5 % 1.5 mM 0.2 mM | Vol (for 1 l) 20 ml 250 ml 1.5 ml 0.4 ml to 1 l | |
| High Salt Buffer: Tris, pH 7.3 Glycerol MgCl2 EDTA pH 8 KCl water | Stock 1 M 50 % 1 M 0.5 M 3 M | Final Conc 20 mM 12.5 % 1.5 mM 0.2 mM 1.2 M | Vol (for 1 l) 20 ml 250 ml 1.5 ml 0.4 ml 400 ml to 1 l | |
| Hypotonic Buffer (HB): Tris, pH 7.3 KCl MgCl2 water | Stock 1 M 3 M 1 M | Final Conc 10 mM 10 mM 1.5 mM | Vol (for 1 l) 10 ml 3.34 ml 1.5 ml to 1 l | |
| 10 x Buffer Tris, pH 7.3 KCl MgCl2 water | Stock 1 M 3 M 1 M | Final Conc 30 mM 140 mM 3 mM | Vol (for 1 l) 30 ml 46.6 ml 3 ml to 1 l |
Cold room set up:
thaw 0.2 M PMSF
prepare hypotonic buffer (500 ml HB + 0.35 ml beta-ME + 0.5 ml 0.2 M PMSF)
prepare freezing solution (20 ml TGME + 0.4 ml DTT, in a 50 ml tube)
prepare tubes
Preparation of Cells
Preparation of Cells
(Note: keep tubes on ice between centrifugations)
Pellet cells at 1500 x g for 8 mins in appropriate tube (eg 50 ml tubes)
Remove supernatant and wash cells once in PBS
Centrifuge at 1000 x g for 10 mins
Remove all supernatant and record the packed cell volume (PCV) in each tube
Fill tube with HB and carefully resuspend cells
Spin at 1000 x g for 5 mins
Verify that the swollen cell volume (SCV) is greater than PCV
Carefully pour off supernatant (pellet may be loose)
Add 2 x PCV volume of HB and carefully resuspend the cells
Place on ice for 10 mins
Cell homogenization
Cell homogenization
homogenize cells by douncing 15 times (for HeLa S3, to be determined for other cell types), check for lysis by microscopy
spin homogenized cells at 2600 x g for 15 mins
record nuclear pellet volume (NPV)
remove supernatant (cytoplasm)
Nuclear Extraction
Nuclear Extraction
calculate NPV/2 and add the amount of low salt buffer and high salt buffer to separate tubes
to each buffer add 0.0007 x NPV/2 of beta-ME, 0.01 x NPV/2 of 0.2 M PMSF, mix well
resuspend nuclear pellets in ½ low salt buffer and dounce 6 times
add the high salt buffer while mixing continuously
mix for 30 mins after the addition of all of the high salt buffer
spin at 15000 x g for 30 min
recover supernatant
for storage, snap freeze in liquid N2 and keep at -80°C