Fig. 3.

PCA of full-length synφX molecules. The first stage of PCA (50-μl reaction volume) was carried out for 35 cycles with 0.2, 0.5, or 1 μlofthe Taq ligation product. PCA products were analyzed on a 0.8% E-gel. (A) Lanes 1 and 6, 1-kb ladder; lane 2, 0.5 μlof Taq ligation product; lane 3, 2 μl of the 0.2-μl PCA; lane 4, 2 μl of the 0.5-μl PCA; lane 5, 2 μl of the 1-μl PCA. The second stage of PCA was for an additional 35 cycles in five new 50-μl reactions. For reaction 1, the 0.2-μl first-stage reaction was continued without change for another 35 cycles with the addition of 0.5 μl of fresh HF polymerase mixture. For reactions 2 and 3, 10 and 20 μl of the 0.5-μl first-stage PCA product was used. For reactions 4 and 5, 5 and 10 μl of the 1-μl first-stage PCA product was used. Analysis was on 0.8% E-gels. (B) Formamide-denatured DNA. (C) Native DNA. (B and C) Lanes 1 and 7, 1-kb ladder; lane 2, 2 μl of reaction 1; lanes 3 and 4, 2 μl of reactions 2 and 3; lanes 5 and 6, 2 μl of reactions 4 and 5. (D) PCR amplification of the products of the second set of PCA products as shown in B and C.(E) Taq ligase assembly of 259 oligonucleotides. A 0.5-μl sample of the ligation products was analyzed on a 2% E-gel (Invitrogen) in duplex form (lane N). One microliter of the ligation products was mixed with 20 μl of formamide, heated to 95°C for 2 min, and then analyzed (lane D). Denatured standards run approximately the same as native standards, based on other experiments (data not shown).