Ribosome Profiling Buffers

Wiki>Fried Lab Ribosome Profiling Buffers

10X MgCl2 Lysis Buffer – 500 mL

200 mM Tris pH 7.5                                          100 mL of 1 M stock

1.5 M MgCl2                                                        152 g

1 M NH4Cl                                                           125 mL of 4 M stock

50 mM CaCl2                                                      25 mL of 1 M stock

4% Triton X-100                                                 20 mL

1% NP-40                                                             5 mL

0.2 U/mL DNase I                                             1 mg — don’t add until use

10 mM PMSF                                                     871 mg — don’t add until use

Do not autoclave.

Top up with MPW to 500 mL.

(Make stock of 4 M ammonium chloride with 106.982 g in 500 mL [endothermic]).

If using 50 mL for a given experiment, add 1 mg DNase I and 87 mg PMSF.

Pelleting Buffer—200 mL

1.1 M sucrose                                                    75.303g

20 mM Tris pH 7.5                                             4 mL of 1 M stock

500 mM NH4Cl                                                  25 mL of 4 M stock

10 mM MgCl2                                                    2 mL of 1 M stock

0.5 mM EDTA                                                     0.2 mL of 0.5 M stock

Resuspension Buffer—50 mL

10 mM Tris pH 7.5                                         0.5 mL of 1 M Tris pH 7.5 stock

10 mM MgCl2                                                 0.5 mL of 1 M stock

60 mM NH4Cl                                                 0.75 mL of 4 M stock

0.5 mM EDTA                                                  ????

-first time that we do this, check the pH first.  It should be 7.5

-add NH4OH to a final concentration of 1 M.  See the pH again. (want it to be ~7.8)

– 1 h at 42 C with 1400 rpm agitation

– filter over 100K MWCO centrifugal concentrators

– add 400 uL of resuspension buffer to the top, mix it up again, and spin again to get more material

-Add 1 M imidazole pH 7 to a final concentration of 20 mM.

(Make a 1M stock of imidazole with 68.077 g in 1 L with 0.5 M HCl to pH).

Nickel NTA Column Elution Buffer—50 mL

20 mM Tris pH 7.5                                                                1 mL of 1 M Tris pH 7.5

300 mM imidazole pH 7                                                      15 mL of 1 M imidazole pH 7

100 mM NaCl                                                                         1 mL of 5 M NaCl stock

Nickel NTA Column Wash Buffer—200 mL

20 mM Tris pH 7.5                                                                4 mL of 1 M Tris pH 7.5

20 mM imidazole pH 7                                                         4 mL of 1 M imidazole pH 7

100 mM NaCl                                                                         4 mL of 5 M NaCl stock

1 M imidazole pH 7–200 mL

200 mmol imidazole = 13.6 g

100 mmol HCl = 8.3 mL of concentrated HCl