CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA 18, January 150 Dear Lu, Please pardon this long delay in answering your letter. I've been traveling since be- fore Christmas. Last week 7 visited Hashingtan University and was qdite impressed with the present nerfnrmance of Kornberg's system. Howard Schactman has been there and has found that the synthesized DI\IA resembles the primer in regard to sedimentation constant and(more significantly) fritrinsic viscosity. hen sonicnted primer is used, the product DhM seems to be shortened accordingly although this lzst result is preliminery according to iloward. The system makes 10x increase in DFlA over primer, but if T.P. is used as primer the transforming activity goes neither up nor down. However, if any one of the nucleotide triphosphates is omited, the transforming activity is nearly completely eliminated in the experimental time period. They blame thk inability to make net increase of T.P. on nucleases known to be present and are according- ly seting about the preparation of really pure enzyme from several hundred pounds of Coli ' vrell*as The system puts in deoxy-WE.: 1 as thym'ine TP and deoxy-inosine TP for GTP in accord with NC pairing expectation Ribonucleotides won't go in. The bqse composition of DNY4 made with Coli primer resembles Coli DXA&hile T2 primer makes ~i!d_ TZ-1Lke pro 1 duct Df.!h. The system seems to be ' well eno:l.gh established to justify using heavy nitrogen DPIA for primer and looking for half-hea1.y molectlles in CsCl gradient, I' 11 make heavy 1'4 D?,IA for them and perh:?ps centrifu.ce it too if they don't :>refer to do it there. !:`e have not dr:ne any new centrifuging(not even to repeat the transfer exnerinent) for tl)ro rriasons. First, l.:relre all out of CsCl CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA and our old sur:pli "r has va.nished, (FIe does not azsy:er letters and his `phone in I-hila. has been disconnected. > We have contracted with a local firm tG make it for us but they won't have any for perhaps another mont& The second difficulty Frank and I have had is th2t we have almost no time on the centrifuge schedu 1 es. We had tied up a maciline for almost a yeat: and now Dintzis, Vinograd, and Sinsheimer quite understandably want to get on with their own work. Our long equilibrium rund require a machine of our own. Accordingly :i:iax has very quickly arranged for us to buy one to be kept in the ph::.ge group.It will be here in about a month. `#hen tvetre going again, weId be glad and interested to run Pl and the antigenicity nutar,ts too. 1 hope we could be ready r.vi thin two months. .we will be if there are no snag8 in setting 112 the machine. To make runs yourself, choose a (3sCl solution of density egual to that of the phage under' the assumption that the density of the phage is the arithmetic average of the protein(l,3> and the DNA(l.70) densities weighted according to the per cent DFJA in a phage, Buffer at pH 7 with 0.01 M phosphate. In the first runs put in enough phage so the CD at 26OA of the starting solution is about 0.4. With this much phage you wi 11 see which way they move incase the band is off scale. You can tell how much to shift the density of the starting solution if/$+$ to put the band in the middle with the aid of the a relation density kfgradient = 8x10' pKeate with w in radians per second and r, the distance in cm from the center of rotation, I suggest a speed of 30-40 thousand rpm for exploratorv runs. The CsCl equilibrium takes about 7 hours and with molecules as big as Fl t%ro or three hours more will be enough to get quite near equilibrium at the above speeds. At lower speeds, the CsCl equilibrium takes still 7 hours but the macromolecules band much more slow1 g.The log w rake of band formation goes as over w . The ratio of senaration between tz tvJo bands to their half-width is& soeed independent So resolution iS the same a.h any s$eed. GOON luck,