Cycle sequencing

Fig. 15. Four-bed PSA system cycle sequence chart (64). EQ, equalization C D A, cocurrent depressurization C D T, countercurrent depressurization R, repressurization A, cocurrent flow T, countercurrent flow. Courtesy of American Institute of Chemical Engineers.

The comparison made in Section 6.1 demonstrates the important effect the amount of purge has on the performance of the carbon canister in terms of limiting the amoimt of HC release. This effect is also shown in the data presented in Fig. 21. In this example, the vehicle has been subjected to the same test cycle sequence as before, but in this case two different levels of purging are examined. Also, a two liter canister is used on the vehicle for the testing at both purge levels, in order to see the effect of purge level on a single canister volume. 

Figure 13. Cycle sequence of a rotary drum filter.

Sequencing of parasite genes provides a powerful tool for the accurate identification of parasites and for systematic studies (Johnson and Baver-stock, 1989 Reddy, 1995 McManus and Bowles, 1996), and is based on either of the two original protocols (Sanger et al., 1977 Maxam and Gilbert, 1980). Cycle-sequencing (Murray, 1989), a PCR-based modification of the... 

Pressure swing adsorption cycle sequence for a four-bed system. (Adapted from Miller, G.Q. and Stocker,., Selection of a Hydrogen Separation Process, UOP Report, January 1999, available at http //www.uop.com/ objects/SelOfHydroSepProc.pdf Cassidy, R.T., Adsorption and ion exchange with synthetic zeolites, in ACS Symposium Series, ed. W.H. Flank, American Chemical Society, Washington, Vol. 135, p. 275,1980.)... 

Yamakawa, H., and Ohara, O. (1997). A DNA cycle sequencing reaction that minimizes compressions on automated fluorescent sequencers. Nucleic Acid Res. 25(6), 1311-1312. 

BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) Store at -20°C. 

The control systems of the cell cycle ensure that the various phases are executed completely and in the correct sequence. Entry into a new phase can only take place when the preceding phase has been completed. In addition, the system allows coupling of processes that are not adjacent in the cell cycle sequence. Thus, there are close mutual regulation mechanisms between M and S phase. Examples of the various control mechanisms are shown in Fig. 13.3. 

Cycle Sequencing of PCR-Amplified Genomic DNAwith Universal Primers... 

Universal recognition sequences must be present in PCR products that are cycle sequenced with universal primers. Many laboratories routinely use universal M13 and M13 reverse tails for their forward and reverse PCR primers, respectively. We have opted for the M13 (but not M13 reverse) sequence in conjunction with a PI72 sequence derived from the Bluescript SK vector. The choice of universal primers was influenced by their estimated free energy of annealing and their melting temperature... 

PCR product purification kit (see 8.2.3.6 Cycle Sequencing of PCR-Amplified Genomic DNA with Universal Primers, subheading Specimen ). 

A thermal cycler (see 8.2.3.2 Polymerase Chain Reaction) is required for the cycle sequencing reaction. For fluorescent cycle sequencing we recommend instruments from Applied Biosystems (e.g. the 16-capillary ABI PRISM 3100 Genetic Analyser) other instruments are available from GC Healthcare and Beckman. For detailed instructions, refer to the respective user s manual or chemistry guide. 

Cycle sequencing products need to be purified, for example by ethanol precipitation prior to loading onto the sequencer. For this purpose, 80 pi water and 10 pi of 3 M sodium acetate are added to each tube or well, and the DNA is precipitated with 250 pi of ethanol. Incubate for 15 min at room temperature and spin for 20 min at maximum speed. The supernatant is removed carefully and 250 pi of 70% ethanol is added. Spin again for 15 min at maximum speed. The pellet (unusually not visible) is air-dried and may be resolved, for example in 20 pi Hi-Di formamide for transfer to the sequencing instrument. For detailed instructions, refer to the respective user s manual or chemistry guide. 

Cycle sequencing of PCR-amplified genomic DNA with universal primers recognises all DNA alterations confined to the sequence between the two PCR primers used for the initial amplification reaction. Other mutations, such as mutations involving the PCR primer sites or large genomic alterations, are not usually recognised. 

A cycle sequence chart for a PSA system incorporating these innovations is shown in Figure 1. The gas for pressurization of adsorbers is transferred via equalizations in pressure between the bed supplying the gas and the bed receiving it. The system is arranged so that one adsorber is always "on-line" supplying product gas. 

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