By M. Demerec (Ed.)
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Extra info for Advances in Genetics, Vol. 1
TLsample as above every minute for 6 mm. Heat the samples for 10 mm at 65°C then cool to room temperature (see Notes 6,7, and 8). 4. Blunting the DNA Two methods may be usedto generateblunt ends for ligation. The use of mung bean nucleaseis more rapid than the method with Exo VII, but has the disadvantage of causing occasional loss of the primer site due to “nibbling-back” or destruction of DNA caused by digestion from nicks or through an excessive ratio of enzyme to substrate. Use of Exo VII has the additional advantage that up to five times more colo- nies are obtained per Itg of DNA than in the nuclease S 1 method.
Filamentous bacterial viruses. Bacteriol Rev. 33, 172-209. 2 Bankier, A. and Barrell, B. G. , Fntsch, E. , and Maniatis, T. ” In Molecular Closmg, A Laboratory Manual. , Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, NY, pp 4-29,4-30. &4PTER 6 Ml3 Phage Growth and DNA Purification Using 96 Well Microtiter Trays Alan l! Bankier 1. Introduction The growth and purification of M 13 DNA from small volume (1 SmL) cultures is a rapid and easily performed procedure (1). The samples can be processed in microcentrifuges in disposable polypropylene tubes and yield sufficient, pure single-stranded DNA (4 M) for five or more sequencing experiments.
2% bacto-agar. To pour plates, melt agar by boiling or microwavmg, cool to around 50°C and pour mto Petri-dishes on a level surface (15-20 ml/plate). Once agar has set, dry plates by storing inverted at 37°C for several hours before use. d. 8% bacto-agar. Melt agar as above and hold at 48OC m a waterbath until needed. 3. Sterile, detergent-free 1-L conical flasks fitted with porous tops or cotton-wool bungs and an orbital incubator capable of shaking these vigorously (around 250 rpm) at 37°C. 4.
Advances in Genetics, Vol. 1 by M. Demerec (Ed.)