After setting clear objectives of what your PCR reaction must accomplish, check that you have the adequate resources. This includes not only budget but also head count, skill level, time equipment, sequence information, sample supply, and other issues If time is most critical, then you may require a colleague's assis- tance or a new instrument to do the project as quickly as possible In a similar token, if the sample is difficult to obtain in abundance the choice of PCr that minimizes the sample requirement becomes more important Selecting one PCR strategy that optimally satisfies every research need is unlikely. At this early planning stage, a compro- mise between competing needs will likely be required. Remem ber that after all the planning is complete, the final PCR strategy still has to evolve at the lab bench Identify Any Weak Links in Your PCR Strategy A There are many parameters that affect the outcome of a PCR action some examples are as follows: PCR reaction chemistry(enzyme, nucleotide, sample, primer, buffer, additives) PCR instrument type(ramp time, well-to-well homogeneity, capacity to handle many samples) Thermal cycling conditions (two-step three-step, cycle segment length-1. e, denaturation, annealing, and exten sion--ramp time, etc. Sample collection, preparation, and storage (DNA, RNa microdissected tissue, cells, and archived material) PCR primer design Detection method (simultaneous detection, post PCR detection). Analysis method(statistical analysis) Like the weakest link in a chain, your final result will be limited by the parameter that is least optimum. For example, suppose that you're studying the tissue-specific regulation of two mRNA forms Regardless of the time spent optimizing the PCr reaction, instru- ment type, and everything to near-perfection, the use of agarose gel electrophoresis may not allow you to reach the conclusion that there are two different mRNA forms if their molecular weight are similar. You might require a separation technique with greater resolving power Suppose that your objective requires quantitative PCR. RNA from 30 samples is collected and RT-PCR is performed. The PCR reaction is run in duplicate and repeated twice on two different PCRAfter setting clear objectives of what your PCR reaction must accomplish, check that you have the adequate resources. This includes not only budget but also head count, skill level, time, equipment, sequence information, sample supply, and other issues. If time is most critical, then you may require a colleague’s assis￾tance or a new instrument to do the project as quickly as possible. In a similar token, if the sample is difficult to obtain in abundance, the choice of PCR that minimizes the sample requirement becomes more important. Selecting one PCR strategy that optimally satisfies every research need is unlikely. At this early planning stage, a compro￾mise between competing needs will likely be required. Remem￾ber that after all the planning is complete, the final PCR strategy still has to evolve at the lab bench. Identify Any Weak Links in Your PCR Strategy There are many parameters that affect the outcome of a PCR reaction. Some examples are as follows: • PCR reaction chemistry (enzyme, nucleotide, sample, primer, buffer, additives). • PCR instrument type (ramp time, well-to-well homogeneity, capacity to handle many samples). • Thermal cycling conditions (two-step, three-step, cycle segment length—i.e., denaturation, annealing, and exten￾sion—ramp time, etc.). • Sample collection, preparation, and storage (DNA, RNA, microdissected tissue, cells, and archived material). • PCR primer design. • Detection method (simultaneous detection, post PCR detection). • Analysis method (statistical analysis). Like the weakest link in a chain, your final result will be limited by the parameter that is least optimum. For example, suppose that you’re studying the tissue-specific regulation of two mRNA forms. Regardless of the time spent optimizing the PCR reaction, instru￾ment type, and everything to near-perfection, the use of agarose gel electrophoresis may not allow you to reach the conclusion that there are two different mRNA forms if their molecular weights are similar.You might require a separation technique with greater resolving power. Suppose that your objective requires quantitative PCR. RNA from 30 samples is collected and RT-PCR is performed. The PCR reaction is run in duplicate and repeated twice on two different PCR 295