Required Solutions

Uniformed Services University

Department of Microbiology and Immunology

Used by the Laboratory of William C. Gause, Ph.D.

Required Solutions

10X TBE: (1 Liter)

TRIS Base 108.0 g, Boric Acid 55.0 g, 0.5 M EDTA 40.0 m

20X  SSPE: (4 Liters)

NaCl   701.2 g, NaH2PO4­H2O   110.4 g, EDTA   29.6 g, *NaOH   34.0 g

*Dissolve reagents in 3200m LdH2O; adjust pH to 7.4;  bring volume to 4 L.

  1X Denaturing Solution: (2 Liters)        1X Neutralizing Solution:  (2Liters)

NaCl 175.32 g                                                  NaCl 175.32 g

NaOH 40.0 g                                                    TRIS HCl 315.02 g

pH should be ~13.0.                                                Adjust pH to 7.5.

1X Prehybridization Solution:  100mL         1X Hybridization Solution:  100mL

(without salmon sperm DNA)                           (without 32P-labeled probe)

20X SSPE 30mL (final = 6X)                             20X SSPE 30mL (final = 6X)

100X Denhardt’s Soln 10mL (final = 10X)          10% SDS 10mL (final=1X)

10% SDS 10mL (final = 1X)                             dH2O 60mL

dH2O 50mL                                                     Need 10mL/blot; used at 49°C; should be

Need 10mL/blot; used at 42°C; should be              kept frozen between assays.

kept frozen at –20°C between assays.

1X Probe Buffer:  100mL                           1X Probe Mix: (27mL)

(make fresh each time)                              Probe Buffer 2.7mL

1.0M TRIS, pH 7.6 50mL                            Oligonucleotide Probe (0.2 mg/mL) 2.0mL

2.0M MgCl2 5mL                                       T4 Phosphonucleotide Kinase(PNK) 1.0mL

0.5M Dithiothreitol (DTT)10mL                   dH2O 11.3mL

dH2O 35mL                                               ₃₂P-ATP 10.0 L ­­ 

6X Wash Solution:  600mL                                High-Stringency Wash:  600mL

20X SSPE 180mL (final = 6X)                                20X SSPE 30mL (final = 1X)

10%SDS 12mL (final = 0.2%)                               10%SDS 12mL (final = 0.2%)

dH2O 408mL                                                        dH2O 558mL