How much of each ingredient do I need to make a 1mL lysis buffer solution?
You need to specify, lysis buffer for bacteria or eukaryotic
cell. The most common buffer cocktail for eukaryotic cells is
composed by an hypertonic solution and cell disrupters. To answer
this question the proper amounts of the ingredients for 1 mL would
be:
8.77 mg of NaCl (150 mM, the hypertonic component)
10 microliters NP-40 [can be replaced by 1 microliter (0.1%) of
Triton X-100, a detergent]
6.06 mg of Tris in 100 microliters [Tris hydroxymethyl
aminomethane, make 10 mL of a 100X solution (that is, 605.5 mg in
10 mL water, adjust to pH 8.0)]
890 microliters of distilled water.
In some cases can be added some protease inhibitors such as
PMSF, leupeptin, Aprotinin, etc. at concentrations of 1
microgram/mL. Store at 4oC for one month.
Now, to make a lysis buffer for bacteria, the composition is
different. For 1 mL mix:
6.06 mg of Tris in 100 microliters, as before
17.53 mg of NaCl (300 mM)
100 microliters of PMSF [Phenylmethylsulfonyl Fluoride, make 10X
solution (1.742 mg in 10 mL of water)]
980 microliters of distilled water
add aprotinin, leupeptin, and pepstatin at final concentration
of 1 microgram/mL.
Usually, to break the bacteria cell wall it is useful a lab
blender. Disruption of both, eukaryotic and bacteria cells, must be
done in cold conditions, usually on ice-water bath.