注：預變(bian)(bian)性溫(wen)度(du)和(he)(he)時(shi)間(jian)可(ke)(ke)以根(gen)據模板(ban)復雜(za)度(du)進行(xing)調整，如模板(ban)復雜(za)度(du)較(jiao)高(gao)(gao)可(ke)(ke)以提高(gao)(gao)預變(bian)(bian)性溫(wen)度(du)并延長時(shi)間(jian)；對于(yu)長度(du)小于(yu)200bp的擴(kuo)增子(zi)，可(ke)(ke)以使(shi)用快速程(cheng)序(xu)，即(ji)變(bian)(bian)性時(shi)間(jian)3秒(miao)，退(tui)火和(he)(he)延伸(shen)時(shi)間(jian)10秒(miao)；對于(yu)長度(du)大于(yu)200bp的擴(kuo)增子(zi)，請(qing)延長退(tui)火和(he)(he)延伸(shen)時(shi)間(jian)至(zhi)30秒(miao)。對于(yu)高(gao)(gao)GC的擴(kuo)增子(zi)，可(ke)(ke)提高(gao)(gao)退(tui)火延伸(shen)溫(wen)度(du)至(zhi)65℃；循環數設(she)置為40-45可(ke)(ke)滿足絕大多數的反應；對于(yu)Tm較(jiao)低的引物，可(ke)(ke)使(shi)用三(san)步法，比(bi)如退(tui)火溫(wen)度(du)設(she)置在(zai)55℃，延伸(shen)溫(wen)度(du)設(she)置為72℃，信號采(cai)集設(she)置在(zai)延伸(shen)步驟。

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