Method: Gel Assay to Determine DNA Content of Phage Lysates

Srini Ramachandra

Purpose:

A crude lysate gel assay can be performed to roughly quantitate the DNA in lysates. This is often a valuable time saving step to determine if the phage yield is sufficient to warrant continuing the procedure.

Note:

Although plate lysates assayed by this method usually give a clear lambda DNA band on a gel occasionally there may be enough bacterial DNA in the lysate to interfere with visualiziation of the phage band. The bacterial DNA in the sample can be degraded with DNaseI prior to adding the dye mix as follows: add 1-2 ul of 0.1 ug DNAseI/ml (fresh dilution) and incubate for 30 minutes at 37 degrees C. This step is always necessary when liquid lysates are assayed.

Time required:

2 hours

Procedure:

1. To 10 ul of lysate add 4 ul of 2.5X SDS-EDTA dye mix (recipe is in the lambda media and solutions section).
2. Heat sample to 65 degrees C for 5 minutes and load onto an agarose gel. Also load three lambda DNA concentration standards of 10 ng 50 ng and 100 ng. Run the gel until the 2 dyes have separated about 1 cm stain and photograph.
3. Compare the lysate sample to the standards and estimate the amount of DNA. Good plate lysates usually have 50-100 ng DNA/10ul. Good liquid lysates have 10-50 ng DNA/10ul.

   Example:

   By the gel assay 10 ul of lysate has 50 ng DNA. If 3.0 ml of the lysate is processed and if we assume a 70% recovery of DNA in the 20 ul final volume the result will be 525 ng DNA/ul TE.

References:

Arber W. Enquist L. Hohn B. Murray N. and K. Murray. (1983). Experimental methods for use with lambda . In: Lambda II (Eds.: R. Hendrix J. Roberts F. Stahl and R. Weisberg) pp. 433-466. Cold Spring Harbor Laboratory Cold Spring Harbor New York.

Helms C. Dutchik J.E. and M.V. Olson. (1987). "A lambda DNA protocol based on purification of phage on DEAE cellulose". In: Meth. Enzymol. 153: p 69-82. Eds.:R. Wu L. Grossman. Academic Press.

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