By Stefan Surzycki
This laboratory handbook supplies an intensive creation to trendy, rate effective suggestions in Molecular Biology, that can locate program in lots of assorted fields. it's the results of useful event, with every one protocol having been used commonly in undergraduate classes and workshops or demonstrated within the author's laboratory. therefore, also they are more likely to paintings in green fingers the 1st time they're performed.
step by step protocols and functional notes are supplied. The distinguishing characteristic of this guide in comparison to others is the specific rationalization of the theoretical mechanisms of every step and the dialogue of the significance of the thoughts. additionally, each one protocol comprises a sign of the time and rate inquisitive about its software. this information will allow the clients to layout their very own variations or to evolve the tactic to diverse structures.
Dr. Surzycki has been educating undergraduate classes and top workshops in introductory Molecular Biology for a few years, in which time he has broadly changed and sophisticated the concepts he has defined during this manual.
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Extra resources for Basic Techniques in Molecular Biology
Table 4. 62 One cannot use equation 1 to calculate DNA concentration precisely when protein is present in the sample since this equation is valid only when the A260/A280 ratio is 2. However this calculation is possible at any A260/A280 ratio using the following equation (derived from: Kalb and Bernlohr, 1977). lg; A260 and A280are absorbance of DNA sample at 260 nm and 280 nm, respectively and,c260 and C280 are absorption coefficients for DNA at 260 nm and 280 nm, respectively. Substituting values of absorption coefficients for DNA and protein from Table 4 gives; N (ug ml- 1 ) = 70A260 - 40A280 (4) A better indicator of protein contamination in DNA samples is the ratio of A260/A234 • DNA has an absorbance minimum at 234 nm, and protein absorbance is high due to the absorption maximum for peptide bonds at 205 nm (Scopes, 1974; Stoscheck, 1990).
12. Combine the DNA from both tubes into one microfuge tube using a cutoff micropipette tip. Combined volume should be about 300 Ill. 13. Add 10 III of DNase-free RNase A and 1111 of RNase T1 and mix by inverting the tube 2 to 3 times. Place the tube into a 37 °Cwater bath and incubate for 30 minutes. 14. 5 M ammonium acetate (half the volume). 15. Add 900 III of 95% ethanol (2 x total volume). Mix well by inverting the tube 4 to 5 times and centrifuge for 10 minutes at room temperature. Pour off the supernatant using the technique described in step 8.
Incubate the solution for 10 to 20 minutes in a 65 °C water bath to dissolve the pelleted DNA completely. Store the DNA sample at-70 -c. 47 48 STE FAN SURZYCKI Small scale protocol 2 1. Use between Ixl G" and lx10 7 cells for a single preparation (see Table 2, Chapter 1 for details of cell weights and expected yields) . To collect cells grown in monolayer culture, add 1 ml of reagent per 10 em? of culture plate area. Do not trypsinize the monolayer cells. 5 ml of cells. Centrifuge the sample at room temperature for 5 minutes at 500 x g.