Formulations of Commonly Used Buffers and Media:

    These are the formulations of buffers we have successfully used over the years. If they are not the same as other formulations, the differences are probably not significant.

Citric Saline:
To make 500mL of 10X solution
    50g KCl
    22g Sodium Citrate
Dissolve in distilled water and bring to 500mL. Sterilize by autoclaving.

    Dilute to 1X with sterile distilled water and use to remove adherent cells from tissue culture dishes. To do this, aspirate off culture media and replace with 1X solution. Let cells sit in the incubator at 37°C, and then monitor cells with microscope: generally they are no longer adherent after as little as 5 minutes. Collect cells by centrifugation, replace citrate saline with regular culture media, and replate cells. Cheap and Efficient!

Mounting Media for Immunofluorescence Microscopy:
To make 50mLs of mounting media add 40g glycerol to 5mLs of 10X PBS and make up to 50mLs volume. To stain for DNA add Hoechst 33258 dye to a final concentration of 2.5mM. If you put the Hoechst dye into your media, be very careful, since this dye is a DNA intercalating agent which may well be carcinogenic.

Phosphate Buffered Saline:
To make 1L of 10X solution
    2g KH2PO4 Potassium dihydrogen phosphate, a.k.a. potassium phosphate monobasic
    14.1g Na2HPO4 Anhydrous sodium phosphate, a.k.a. sodium phosphate dibasic.
    2g KCl Potassium Chloride
    80g NaCl Sodium Chloride
pH to 7.4 with 5N NaOH. (be careful, concentrated NaOH, perhaps surprisingly given that every lab has some around, is quite dangerous. It can blind you if it gets in your eyes!)

    1X PBS is 1.47mM KH2PO4, 10mM Na2HPO4, 2.7mM KCl, 137mM NaCl pH=7.4. It is a more or less physiological buffer which living mammalian cells can tolerate at least for a short time. It is frequently used to wash cells prior to protein extraction or immunostaining. It is also useful for antibody incubations in immunocytochemical staining of cells in tissue culture and sections. One caveat is that phosphatase based detection systems are inhibited by high concentrations of phosphate, the phosphatase reaction product, so you need to do a wash in some sort of phosphate free buffer before you use these. For antibody staining and washing you can also add 0.1% Tween 20 (Polyoxyethylene 20-sorbitol monolaurate) or 0.1% Triton X-100 non-ionic detergents to reduce background staining.

Tris Buffered EDTA (TBE):
To make 1L of 10X solution
107.8g Tris base
~55g Boric acid
7.44g EDTA
add less than total amount of Boric acid, dissolve this and the Tris in 800 ml of distilled water and make pH to 8.3 by adding more boric acid. Finally make to 1L final volume.
This is the standard buffer for running DNA in agarose gels.

Tris Buffered Saline:
To make 1L of 10X solution
    12.1g Tris base
    87.66g NaCl
pH with concentrated HCl (be careful!) to 7.5

    1X TBS is 10 mM Tris/HCl, 150 mM NaCl, pH=7.5. We use TBS routinely for ELISA, immunoblots etc. Can also add 0.1% Tween 20 or 0.1% Triton X-100 non-ionic detergents to final concentration of 0.1% reduce background.

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