Page Title

Q-R

P rotocols Q-R

Running Buffer (SDS-PAGE)
5X Running Buffer:
144 g Glycine
60 g Tris Base
QS to 1 L with dH2O

1X Running Buffer:
1L 5X Buffer
25 ml 20% SDS
QS to 5 L with dH2O

RF1 (for competent cells)
              100 ml          250 ml
100 mM RbCl             10 ml                 25 ml
50 mM MnCl_4H2O   5 ml               12.5 ml
30 mM KOAc               3 ml                 7.5 ml
10 mM CaCl2_2H2O 60 ml             150 ml
pH to 5.8 (do not add NaOH)
After pH, QS to 81.25 ml or 203.1 ml,
respectively.
Sterile filter and add sterile 15%
glycerol(18.75 ml and 46.9 ml, respectively).

RF2 (for competent cells)
                            100 ml
10 mM MOPS                    10 ml   of 0.1 M
10 mM RbCl                       1 ml of 1 M 1M
75 mM CaCl2_2H2O        7.5 ml of 1 M
15% Glycerol                     18.75 ml of 80% stock
dH2O                                    62.75 ml (add 60 ml
first, pH to 6.8 and QS to 100 ml final vol.
-Sterile filter.

Reducing Sample Buffer (4X, SDS-PAGE)

Reagent                                                                    Amount
Tris-HCL (MW 157.6)                                               3.152 g
(Dissolve in 45 ml water, pH to 6.8, QS to 50 ml)
SDS                                                                              8.0 g
Glycerol                                                                      40 ml
Bromphenol Blue                                                     0.1 ml
DTT                                                                          0.62 g (40 mM)
QS to 100 ml. Aliquot into 1 ml epi tubes.

For non denaturing omit adding SDS.

RBC Lysis Buffer
1-Tris (hydroxymethyl)
aminomethane (0.17 M) 20.6 g/L

2-Amonium Chloride (0.16 M) 8.3 g/L

Add 10 ml of Tris aminomethane to
90 ml of Amonium Chloride. pH to
7.2.
Sterile filter.