Answers to Sample Calculations #1

  1. Make a 120 ml agarose gel of 0.7% that is 0.5X TBE. The TBE (Tris, Borate, EDTA) stock is 5X and FW agarose is 210.

    Use 0.84 g agarose and 12 ml 5X TBE and 108 ml H2O.

  2. You have a 50X stock of buffer and you need 150 µl of 1X. How do you make this?

    Use 3 µl of the 50X stock and 147 µl of H2O

  3. You have a DNA solution that is 125 µl; how much ethanol do you add if you add 2X the volume of ethanol?

    To precipitate DNA, you add ethanol. Therefore, add 250 µl of ethanol to the 125 µl of DNA.

  4. Make 500 ml of a 10X TE (Tris, EDTA) stock pH 8.0. 1X TE is 10mM Tris and 1mM EDTA. FW of Tris is 121.1 and EDTA is 187.

    Use 6.05 g Tris
    0.935 g EDTA and water up to 400 ml
    pH this with HCl and add water up to 500 ml

  5. You want to digest 2 µg of DNA with BamH I; your DNA stock is 1.2 mg/ml. Use 1 µl of BamH I and get your final volume to 30 µl. You need to have final concentrations of buffer and BSA (bovine serum albumin) to 1X but their stocks are 10X(buffer) and 100X(bovine serum albumin). Make that mixture.

    24.1 µl H2O
    3 µl 10 X buffer
    0.3 µl BSA
    1.6 µl DNA
    1 µl BamH I (always add the enzyme last)

  6. Make 500 ml the following stock solution: 0.6 M Tris, 60 mM SDS (sodium dodecly sulfate is a denaturing detergent; FW = 257), 1% powdered milk (FW = 49) , 1.3 M NaCl (FW = 58.5).

    measure about 400 ml of water and add:
    36.3 g Tris
    7.71 g SDS
    5 g powdered milk
    38.0 g NaCl
    dissolve and adjust volume to 500 ml

  7. When making a buffer at a certain pH, one starts by dissolving the buffer into water and you read the pH is 5.2. What kind of substance would you add to get the pH to 7.2? And, if you wanted to make exactly 1L of this buffer, what order of what volumes would you add to not exceed the desired volume?

    Start with about 700 ml of H2O
    add the appropriate amount of dry buffer
    dissolve buffer
    pH with base (e.g. 5 M NaOH)
    bring volume up to 1 L with H2O

  8. 1 OD260 unit of a double stranded DNA solution equals 50 µg /ml. You OD a solution and the spectrophotometer reads 0.057 but you are measuring a solution that is 3 µl DNA and 97 µl H2O. What is the concentration of the original DNA stock solution?1.

    With an OD260of 0.057, the measured DNA solution is 2.85 µg/ml.
    But, you have diluted the original stock of DNA 33.3 fold (100 µl total volume divided by 3 µl DNA).
    Therfore, multiply 2.85 by 33.3 and you have the concentration of the original DNA solution; 95 µg/ml.

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