How to make gene clean solutions:
For NaI need:
-powdered NaI, 99.5%, A.C.S. reagent (from Sigma) (MW=149.89 g / mol)
-100 mM sodium thiosulfate (Na2S2O3)
-MW=248.18 g / mol
-Add 2.48 g to a final volume of 100 mL dH2O
-Autoclave
-10 X Tris-acetate-EDTA (pH 7.2) (TAE)
-1 M Tris-acetate (pH 7.2)
-MW=121.1 g / mol
-Add 12.1 g of Tris base to 80 mL dH2O, pH to 7.2 with glacial acetic acid, then bring up
to final volume of 100 mL.
-Autoclave
-0.5 M EDTA (pH 8.0)
-MW=372.24 g / mol
-Add 18.61 g of EDTA to 70 mL dH2O, pH to 8.0 with NaOH (either pellets or
concentrated solution).
-Autoclave.
-10 X TAE
-Add 50 mL 1 M Tris-acetate (pH 7.2) (final concentration = 500 mM), 20 mL 0.5 M
EDTA (final concentration = 100 mM), and 30 mL dH2O to a bottle.
-Autoclave
-6 M NaI
-Add 90 g of NaI , 10 mL of 10 X TAE (final concentration = 1X), 10 mL of 100 mM Na2S2O3
(final concentration = 10 mM), and 50 mL of dH2O to a 100 mL bottle. Mix. (Note: Exothermic
reaction between NaI and water so solution will heat up.; To dissolve NaI, let solution sit and
swirl on occasion until all NaI is in solution.) Bring up to final volume of 100 mL. Wrap bottle
in foil. Store at 4C.
-For silica need:
-3 M NaI
-Add 11.24 g of NaI , 2.5 mL of 10 X TAE (final concentration = 1X), 2.5 mL of 100 mM
Na2S2O3 (final concentration = 10 mM), and 12.5 mL of dH2O to a 100 mL bottle. Mix. (Note:
Exothermic reaction between NaI and water so solution will heat up.; To dissolve NaI, let
solution sit and swirl on occasion until all NaI is in solution.) Bring up to final volume of 25 mL.
Wrap bottle in foil. Store at 4C. (Note: Enough to make two batches of silica)
-Phosphate-buffered saline (PBS)
-To 400 mL dH2O, add 4 g of NaCl, 0.1 g of KCL, 0.72 g of Na2HPO4, and 0.12 g of KH2PO4.
Mix.
-Adjust pH to 7.4 with HCl. (Note: pH should be close to 7.4 when measured.)
-Bring up to volume of 500 mL.
-Autoclave.
-Silica
-Measure out 1 g of silica into a new autoclaved Oakridge tube.
-Resuspend in 10mL PBS. Let settle for 2-3 hrs. Remove supernate using a glass pipette.
-Repeat above wash in PBS once.
-Centrifuge tubes for 5 mins at 2000 X g (4500 rpm in SS34).
-Remove rest of the supernate.
-Resuspend pellet in 10 mL 3 M NaI.