RFLP of PCR Products

  1. Since pipetters are not accurate at very small volumes, make a mastermix for each enzyme (or sets of enzymes) you are using. For example, a mastermix for ten 10ul PCR samples using a final volume of 15ul is as follows:

    MASTERMIX (10+1 samples)  
     
    10X buffer for the enzyme   1.50 ulx 11 samples  = 16.50 ul
    Enzyme (1/100th of final volume)   0.15 ul x 11 samples = 1.65 ul
    BSA additive (1/100th of final volume)   0.15 ul x 11 samples = 1.65 ul
    ddH2O   3.20 ul x 11 samples = 35.20 ul
        5.00 ul x 11 samples = 55.00 ul
           


    Enzyme notes:

    The enzyme used depends on your particular situation. Refer to other protocols to determine which enzyme to use. Enzyme buffers will differ depending on which enzyme you use. Refer to the enzyme charts on the freezer door to determine which buffer to use. Additionally the enzyme chart will provide information on whether the enzyme you are using needs a BSA additive and what incubation temperature to use
  2. .For each sample, pipette 5ul of mastermix and 10ul of PCR product into a new tube.
  3. Seal tubes and incubate for 1-2 hours at the proper incubation temperature for the enzymes you are using (usually 37°C).
  4. Depending on the fragment sizes expected, RFLP patterns can be visualized using agarose gel (large fragments) or acrylamide gel (small fragments).