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Medical Subject Headings (MeSH):
Restriction Enzymes and the "New Genetics," 1970-1980
September 24, 1975
I have settled in Edinburgh and the family is quite pleased. I am doing some learning experiments with recombination and transfection.
The lab is certainly well equipped for all of the [lambda] work and there seem to be good provisions for EM heteroduplexing.
However, there are no cell culture facilities readily available on this campus. There probably will be cell culture opportunity
in the MRC cytogenetics unit which is across town at Western General Hospital. It will probably take time to work out the
arrangements, and I believe I can grow the SV40 there.
For these reasons, your offer to supply the dl 1009 is not only generous but will greatly facilitate the project. Ken had
planned to talk to you at Madrid, but evidently you were not there at the same time. Could you send the DNA directly to me?
Since spending the day at your lab with you, I have had a chance to go over the papers you gave me and to think about the
project in more detail. I would appreciate it if you would look at the outline of the protocol and see if you think it is
1. Separate the DNA of form I 1009 from the ts helper form I on 1.4 percent agarose/Eth Br. as in Plate III, JMB 89 -- Lai
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2. Localize the Form I dl 1009 c 360 A degrees light and cut from agarose
3. Electrophorese DNA from agarose in dialysis membrane sac.
4. Conc. this DNA by alcohol ppt.
5. Establish conditions for single cut Hind III digestion
6. A) Using these conditions do partial digestion and B) again electrophorese on 1.4 percent agarose/Eth Br.
7. Localize DNA by 360 A degrees light and cut out full length dl 1009 linears. (Standards could be made by limit digestion
of dl 1009 with EcoRI). Separate from 1.4 percent agarose by electrophoresis into membrane sac.
8. This DNA to be used for ligase vacation and transfection
9. Pick plaques and make stocks
10. Prepare DNA from stocks
11. Remove inserted DNA by Hind III digestion and electrophoresis and isolate inserted DNA
12. Do heteroduplex mapping c with SV40 -- wild type
13. Select stocks which has deletion loop .2 units from end Others will be approximately[?] .4 from end
This raises some questions: How much DNA should be applied to gels to end up c 3 [micro]grams for the restriction digestion?
Could you draw a diagram of electrophoresis sac for separating the DNA? Do you think it would be more economical to skip the
2nd electrophoresis (Step 6 B) and use the partial digest directly for recombination? Partial digestion would generate 16
different molecules wheras [sic] selecting for full length linears with dl 1009 would only comprise 4 different molecules.
Thanks for your help. I certainly like the idea for the project and am anxious to proceed.