[박상규] Preparation of Nuclei from mouse Liver

[Anonymous]

Preparation of Nuclei from mouse Liver

 

stocks

1.     2.5 M sucrose (85%,w/v) : to make 1 liter, dissolve 850g of  sucrose in distilled water and adjust to a total volume of 1 liter. Allow a day for solving (at 60 degree) and filtration of the sucrose. Store at 4 degree

2.     2 M KCl (filter) : to make 1 liter, dissolve 149.1 g of KCl in distilled water and adjust to a total volume of 1 liter. Filter and store at 4 degree.

3.     1M triethanolamine –HCl, pH 7.5 (filter) : to make 1 liter, dissolve 185g of triethanolamine-HCl in distilled water, adjust pH with HCl, and dilute to a total volume of l liter. Filter and store at 4 degree.

4.     1M MgCl2 (filter) : to make 1 liter, dissolve 95.2 g of MgCl2 in distilled water and adjust to a total volume of 1 liter. Filter and store at 4 degree.

5.     0.1 M PMSF in ethanol : to make 10 ml, dissolve 174 mg of PMSF in 10 ml of ethanol. Keep on ice. Prepare fresh.

6.     1 M DTT : to make 10ml, dissolve 1.54 g of DTT in distilled water. Keep on ice. The DTT will form a precipitate, which has to be dissolved by moderate heating just before use. Prepare fresh.* you can use 2-mercaptoethanol instead of DTT

 

Solutions

7.     0.25M sucrose, 25 mM KCl, 50mM triethanolamine-HCl, pH 7.5, 5mM MgCl2, 0.5mM PMSF, and 1mM DTT(10mM 2-mercaptoethanol). Keep on ice

8.     2.3M sucrose, 25mM KCl, 50mM Triethanolamine-HCl, pH7.5, 5mM MgCl2, 0.5mM PMSF, and 1mM DTT(10mM 2-mercaptoethanol). Keep on ice

Steps

9.     Trim livers from 5-10g from mouse and chop into small pieces using razor blades.

10.  make a 30% homogenate (15 strokes) in solution 7 using a tight –fitting potter-elvhjelm homogenizer

11.  filter th homogenate through four layers of cheesecloth and add 5ul of solution 6 per ml of homogenate and spin for 15 min at 800Xg

12.  remove the supernatant and homogenize the large and loose pellet in a clean potter homogenizer and dilute to 5ml in solution 7

13.  Add exactly 2 vol of solution 8. mix well and layer on top of 5 ml solution 8 in ultra centrifuge tubes. Fill up the tubes completely and balance. Spin at 124,000Xg for 1 hr at 4 degree.

14.  discard the supernatant and harvest pellet and resuspend the nuclei in 2ml of solution 7.

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