Original Repository: Stanford University Libraries. Department of Special Collections and University Archives. Paul Berg Papers
Reproduced with permission of Paul Berg.
Medical Subject Headings (MeSH):
Protein Synthesis, Tumor Viruses, and Recombinant DNA, 1959-1975
March 15, 1972
I am sorry that it has taken so long to send you this material but Marianne was ill and it took some time for her to get it
I am including the protocol for preparing very highly labeled P32 polyoma DNA. It is obviously applicable to SV40 DNA as well.
I think it is quite straight forward but if there are any questions don't hesitate to let me know.
The two photographs show the data for two different experiments and should give you an idea of the kind of data the method
provides. I think we can further reduce the concentration of P32 DNA to the order of 2-3 x 10 [to the negative 5] A260; that
would increase the sensitivity of detecting unlabeled complementary sequences. The xerox copies show the protocol and data
for Cot-16. I enclose it to illustrate the ingredients of the annealing mixture and the schedule for taking samples used
in that experiment. After the samples are removed from the appealing mixture they are diluted in the S1 enzyme digestion buffer
and frozen. After all the samples are collected, they are incubated with the en5rme at 37 degrees for an hour and then acid
precipitated. The amount of P32 which remains acid precipitable is a measure of the amount of DNA which is annealed. Also
attached is the computer printout for calculating the curves and the Cot 1/2 for the three samples of Cot-16.
More recently, we've handled the data somewhat differently and have plotted it in a linear way. Cot-16 is replotted using
only the time points up to 24 hours or less. You can see the points (unfortunately we did not collect enough early points
because the experiment was not designed for this purpose) yield a nice straight line which enables us to calculate the second-order
rate constant for annealing. These second-order rate constants yield Cot 1/2 values which are in good agreement with the predicted
The advantage of this kind of plot is that we can use the short time intervals, even up to an hour to determine the rate constants.(See
Graph 1). A second experiment using the same technique of handling the data gave equally good results (Graph 2). The set of
computer output shows how the program calculates from the same points what we call a Wetmur Plot. (after Wetmur and Davidson).
The program gives us the values for the theoretical second-order line which best fits the experimental points.
We do have some of the Taka-diastase powder from which the S1 enzyme can be prepared (see enclosed reprint). If you want some
I can send you a little but it is commercially available (Enzyme Development Corporation, 2 Penn Plaza, New York, New York,
We have finally succeeded in making the SV40 [lamda] dvgal hybrid duplex circles; the yield is quite good, about 25%, and
I hope now to make sufficient quantities for some interesting biological experiments. We do have some now and we plan to try
to transfer-3T3 with them. We have also synthesized SV40 dimer circles by fusing monomeric linears and we shall test their
infectivity and transforming activity as well. It should be interesting to make SV40 dimer from viruses having different
genotypes. I am hopeful I can find other interesting DNA's to insert into SV40 that can broaden our usefulness of this
I hope all is well at the Salk. Please give my very best to Maureen, Margarite and all the rest of the people there.