Question

Calculate the volumes needed to prepare the following: The following stock solutions will be provided: 200 mM Tris-Acetate, 200 mM NaCl, 50 mM EDTA, and four stock solutions of 10 μM SSB, 100 μM 8 nt long ssDNA (short ssDNA), 100 μM 20 nt long ssDNA (long ssDNA) and 100 μM 20 nt long complementary DNA. 1. Using the stock solutions, prepare 10 mL incubation buffer (50 mM Tris-Acetate, 50 mM NaCl, 10 mM EDTA, pH 8.2). 2. Prepare 250 μl of 1 μM working solution of SSB from the 10 μM stock. (use this incubation buffer further to prepare all your working solutions) 3. Prepare 200 μl of 1 μM DNA working solutions using the 100 μM of FAM labeled stock solutions provided for short ssDNA and long ssDNA.


Answer 1

1. TAE buffer is commonly prepared as a 50M stock solution for laboratory use. A 50M stock solution can be prepared by dissolving 242g Tris base in water, adding 57.1mL glacial acetic acid, and 100mL of 500mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter. This stock solution can be diluted 49:1 with water to make a 1M working solution. This 1M solution will contain 40mM Tris, 20mM acetic acid, and 1mM EDTA. (For 1M EDTA solution,EDTA.2H2O - 37.22 gm Distilled water - 100 mL)

So we need to take 2.42g of Tris base,0.571mL of glacial acetic acid,0.58g(50mMolNaCl stock soln) NaCl and 0.37g of EDTA shold dissolve 10mL of Water to prepare incubation buffer.