By Robert F. Schleif
This quantity has advanced from a laboratory tools publication that certainly one of us first compiled approximately fifteen years in the past. considering that point the ebook has gone through many minor revisions to be able to contain new equipment and up to date types of older tools. the outcome has been an more and more beneficial and extra generally circulated booklet. in spite of the fact that, the new sequence of technological explosions normally lumped jointly below the identify of "recombinant DNA expertise" has been a turning aspect within the evolution of this formerly underground booklet. Minor revisions will not do. to maintain the ebook worthwhile we now have needed to make significant revi sions and additions. the result's a dramatically extended booklet that are meant to be extra worthy to extra humans. the bigger measurement and wider usefulness of the booklet have made this extra formal ebook appear a cheap step to take. one of many purposes that this quantity could be invaluable to many folks is that it contains simply systems which were used again and again by means of us and that experience confirmed hugely trustworthy either to ourselves and to others in our laboratories.
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Extra resources for Practical Methods in Molecular Biology
01 M CaCI 2• Freshly poured 20-mm-thick plates seem to work a little better than 15-mm plates which have been stored at 4 °C for a few months, but the difference is rarely important. 5. 5-7 h at 37°C until there is confluent lysis of cells. Compare the resulting plate to a plate which received cells but no phage. Sometimes the cells do not appear to have lysed but a high titer will result anyway. 6. 01 M MgS04 • Break up the top agar and scrape it into a Sorvall tube. Add 10 drops of chloroform, vortex, let sit about 5 min, vortex again, and then spin at 10,000 X g for 20 min.
3. Let sit 15 min at 37°C for adsorption, add agar, and then pour. Fresh plates make the plaques easier to see. TRANSDUCING 1. Grow cells overnight to stationary phase in YT medium. 2. 1 ml of cells. 1 ml on selective plates. In 1 to 2 days 100-1000 colonies will appear. Transducing with serial10-fold dilutions of the P1 lysate is the most efficient approach to obtaining an appropriate mUltiplicity of infection. Take a transductant from the plate which received the fewest phage yet also contains at least 10 times as many transductants as revertants.
It has been found that the tail fibers of ¢80 are temperature sensitive, so this phage will not form plaques at 42°C. One of the most popular hosts for growing lambda, the C600 strain of E. coli, is resistant to phage T1, which plagues some laboratories. This bacterial strain is also resistant to ¢80, since these two phage use the same receptor on the cell surface. LARGE-SCALE GROWTH IN LIQUID When it is necessary to isolate more than 1012 phage it is easier to do so by growing the bacteria and phage in liquid rather than on an agar surface.